Abbott RealTime 2N40

HBV 51-608234/R4

quantitation of HBV DNA. The selection of a highly conserved region in the Surface
NOTE: Changes Highlighted gene provides for the detection of HBV genotypes A, B, C, D, E, F, G, and H. The
location of the target region in the N terminal third of the Surface gene ensures that
Customer Service: 1-800-553-7042 the assay is not impacted by YMDD mutants, HBsAg escape mutants, or drug-resistant
This package insert must be read carefully prior to use. Package insert instructions mutants, as this region is essential for the assembly and secretion of subviral particles,
must be followed accordingly. Reliability of assay results cannot be guaranteed if there and tolerates only minor structural changes.10
are any deviations from the instructions in this package insert.
The assay is standardized against the World Health Organization (WHO) International
Key to symbols used Standard for Hepatitis B Virus DNA (NIBSC).11 Results are reported in International Units
per milliliter (IU/mL) or copies/mL.
List Number Calibrator A BIOLOGICAL PRINCIPLES OF THE PROCEDURE
Lot Number Calibrator B The Abbott RealTime HBV assay consists of three reagent kits:

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• Abbott RealTime HBV Amplification Reagent Kit
In Vitro Diagnostic Medical

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Device
Negative Control • Abbott RealTime HBV Control Kit
• Abbott RealTime HBV Calibrator Kit
The Abbott RealTime HBV assay uses PCR to generate amplified product from the

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Store at -10°C or colder Low Positive Control
DNA genome of HBV in clinical specimens. A DNA sequence that is unrelated to the
HBV target sequence is introduced into each specimen at the beginning of sample

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Manufacturer High Positive Control preparation. This unrelated DNA sequence is simultaneously amplified by PCR and
serves as an internal control (IC) to demonstrate that the process has proceeded

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correctly for each sample. The amount of HBV target sequence that is present at each
Consult instructions for use Internal Control amplification cycle is measured through the use of fluorescent-labeled oligonucleotide

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specifically bound to the amplified product. The amplification cycle at which fluorescent
Expiration Date
signal is detected by the Abbott m2000rt is inversely proportional to the log of the HBV
Amplification Reagent Pack DNA concentration present in the original sample.

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CAUTION: Handle human Sample Preparation
sourced materials as The purpose of sample preparation is to extract and concentrate nucleic acid, to
potentially infectious. Consult For Prescription Use Only make the target accessible for amplification, and to remove potential inhibitors

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instructions for use. of amplification from the extract. This process is accomplished by the m2000sp,
(Infection Risk)
an automated sample preparation system designed to use magnetic microparticle

Warning
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processes for the purification of nucleic acids from samples. The assay is suitable for
use with both 0.5 mL and 0.2 mL sample input volumes.
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The Abbott mSample Preparation SystemDNA (4 x 24 Preps) reagents lyse the virion,
capture the nucleic acids, and wash the particles to remove unbound sample
See REAGENTS section for a full explanation of symbols used in reagent component
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components. Proteinase K is included in the lysis step to digest proteins associated with
naming. the nucleic acids.12,13 The bound nucleic acids are eluted and transferred to a 96-deep
NAME well plate. The nucleic acids are then ready for amplification. The IC is introduced into
Abbott RealTime HBV Assay the sample preparation procedure and is processed along with the calibrators, controls,
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and specimens.
INTENDED USE Reagent Preparation and Reaction Plate Assembly
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Abbott RealTime HBV assay is an in vitro polymerase chain reaction (PCR) assay The Abbott m2000sp combines the Abbott RealTime HBV amplification reagent
components (HBV Oligonucleotide Reagent, DNA Polymerase, and Activation Reagent).
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for use with the Abbott m2000 SystemDNA reagents and with the Abbott m2000sp and
m2000rt instruments for the quantitation of Hepatitis B Virus (HBV) DNA in human The Abbott m2000sp dispenses the resulting master mix to the Abbott 96-Well Optical
serum or plasma (EDTA) from chronically HBV-infected individuals. The assay is Reaction Plate along with aliquots of the nucleic acid samples prepared by the Abbott
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intended for use as an aid in the management of patients with chronic HBV infection m2000sp. After manual application of the Abbott Optical Adhesive Cover, the plate is
ready for transfer to the Abbott m2000rt.
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undergoing anti-viral therapy. The assay can be used to measure HBV DNA levels at
baseline and during treatment to aid in assessing response to treatment. The results Amplification
During the amplification/detection reaction on the m2000rt instrument, the target DNA
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from the Abbott RealTime HBV assay must be interpreted within the context of all
relevant clinical and laboratory findings. Assay performance for determining the clinical is amplified by the DNA Polymerase in the presence of deoxynucleotide triphosphates
(dNTPs) and magnesium. First, the HBV and IC primers anneal to their respective
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stage of HBV infection has not been established. Clinical performance characteristics
have been established for individuals treated with adefovir dipivoxil. This assay is targets and are extended by the polymerase. After a denaturation step in which the
temperature of the reaction is raised above the melting point of the double-stranded
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not intended for use as a screening test in blood or blood products for HBV or as a
diagnostic test to confirm the presence of HBV infection. DNA product, the newly created DNA strand is denatured from the target DNA.
During each round of thermal cycling, amplification products dissociate to single strands
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SUMMARY AND EXPLANATION OF THE TEST at high temperature, allowing primer annealing and extension as the temperature is
HBV is a small, circular, partially double-stranded DNA virus of approximately 3,200 lowered. Exponential amplification of the target is achieved through repeated cycling
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base pairs.1 The virus can cause lifelong infection, cirrhosis (scarring) of the liver, between high and lower temperatures. Amplification of both targets (HBV and IC) takes
liver cancer, liver failure, and death.2 The prevalence of HBV infection and the method place simultaneously in the same reaction.
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of transmission vary greatly around the world. In countries with a high prevalence of
chronic HBV infection, the most common route of infection is from mother-to-child at
birth or from child-to-child during early childhood. In areas of low prevalence, infection
is usually acquired during adulthood through intravenous drug use or high-risk sexual
activity.3 The risk of developing chronic HBV infection from acute exposure ranges from
90% in newborns of HBV-infected mothers to 25-30% for children under 5 and less than
10% in adults.1 Immunization is the most effective way to prevent HBV infection, and can
offer greater than 95% protection against the development of chronic infection.3
Quantitation of HBV DNA is important in the evaluation and management of patients
with chronic HBV infection. Current guidelines recommend HBV viral load to determine
which chronic HBV patients should be treated and to monitor their response to
therapy.4,5,6 A low baseline viral load has been shown to be predictive of response to
therapy.5 Conversely, a high baseline viral load is predictive of resistance to therapy
as well as relapse following therapy, and has also been found to be an independent
risk factor for hepatocellular carcinoma.7 Current treatment options include interferon,
peginterferon, and antiviral drugs such as lamivudine, adefovir, and tenofovir.5,6,8
HBV DNA in serum or plasma can be quantitated using nucleic acid amplification
or signal amplification technologies.9 The Abbott RealTime HBV assay uses PCR
technology combined with homogeneous real time fluorescent detection for the
The target sequence for the Abbott RealTime HBV assay is in the Surface gene in the
HBV genome. This region is specific for HBV and is highly conserved. The primers are
designed to hybridize to this region with the fewest possible mismatches among HBV
genotypes A through H.
The IC target sequence is derived from the hydroxypyruvate reductase gene from the
pumpkin plant Cucurbita pepo, and is provided as a DNA plasmid in a buffer solution.
Detection
The presence of HBV amplification products is detected during the extension/anneal
step by measuring the fluorescence of the HBV probe that binds to the target during
the extension/anneal step. Similarly, the presence of IC amplification is detected during
the extension/anneal step by measuring the fluorescence of the IC probe.
The HBV and IC probes are single-stranded DNA oligonucleotides consisting of a probe
sequence with a fluorescent moiety that is covalently linked to the 5' end of the probe
and a quenching moiety that is covalently linked to the 3' end of the probe.
In the absence of the HBV or IC target sequences, probe fluorescence is quenched.
In the presence of HBV or IC target, the HBV or IC probes specifically bind to their
target. During the extension/anneal step, the DNA polymerase cleaves, or nucleolytically
digests, the bound probe as it moves along the template strand. This separates the

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fluorophore from the quencher, allowing fluorescent emission and detection.
The HBV and IC probes are each labeled with a different fluorophore, thus allowing for
simultaneous detection of both amplified products at each cycle. The amplification
cycle at which fluorescent signal is detected by the Abbott m2000rt is inversely
proportional to the log of the HBV DNA concentration present in the original sample.
PREVENTION OF NUCLEIC ACID CONTAMINATION
The possibility of nucleic acid contamination is minimized because:
• The Abbott RealTime HBV assay performs PCR amplification and oligonucleotide
hybridization in a sealed 96-Well Optical Reaction Plate.
• Detection is carried out automatically without the need to open the 96-Well
Optical Reaction Plate.
• Aerosol barrier pipette tips are used for all pipetting. The pipette tips are
discarded after use.
• Separate dedicated areas are used to perform the Abbott RealTime HBV assay.
Refer to the SPECIAL PRECAUTIONS section of this package insert.
REAGENTS
Abbott RealTime HBV Amplification Reagent Kit (List No. 2N40-90)
1. Abbott RealTime HBV Internal Control (List No. 2G34Y)
(4 vials, 0.2 mL per vial)
• Less than 0.01% noninfectious linearized DNA plasmid in a buffer solution with
carrier DNA. Preservatives: 0.085% Sodium azide and 0.15% ProClin® 950.
2. Abbott RealTime HBV Amplification Reagent
Pack (List No. 2N40)
(4 packs, 24 tests/pack)
• 1 bottle (0.078 mL) DNA Polymerase (5.4 to 5.9 Units/µL) in a buffered solution
with stabilizers.
• 1 bottle (0.918 mL) HBV Oligonucleotide Reagent. Less than 0.1% synthetic
oligonucleotides (4 primers and 3 probes), and less than 0.2% dNTPs in a
buffered solution with a reference dye. Preservatives: 0.085% Sodium azide and
0.15% ProClin 950.
• 1 bottle (0.778 mL) Activation Reagent. 38 mM magnesium chloride in a buffered
solution. Preservatives: 0.085% Sodium azide and 0.15% ProClin 950.
Abbott RealTime HBV Control Kit (List No. 2N40-80)
1. Abbott RealTime HBV Negative Control (List No. 2G34Z)
(8 vials, 1.3 mL per vial)
• Negative human plasma found to be nonreactive by FDA licensed tests for
antibody to HCV, antibody to HIV-1, antibody to HIV-2, and HBsAg. The material
is also tested and found to be negative by FDA licensed PCR methods for HIV-1
RNA and HCV RNA. Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950.
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2. Abbott RealTime HBV Low Positive Control (List No. 2G34W)
(8 vials, 1.3 mL per vial)
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• Heat-inactivated plasma reactive for HBV DNA in negative human plasma.
Negative human plasma found to be nonreactive by FDA licensed tests for
antibody to HCV, antibody to HIV-1, antibody to HIV-2, and HBsAg. The material
is also tested and found to be negative by FDA licensed PCR methods for HIV-1
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RNA and HCV RNA. Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950.
3. Abbott RealTime HBV High Positive Control (List No. 2G34X)
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(8 vials, 1.3 mL per vial)
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• Heat-inactivated plasma reactive for HBV DNA in negative human plasma.
Negative human plasma found to be nonreactive by FDA licensed tests for
antibody to HCV, antibody to HIV-1, antibody to HIV-2, and HBsAg. The material
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is also tested and found to be negative by FDA licensed PCR methods for HIV 1
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RNA and HCV RNA. Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950.
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Abbott RealTime HBV Calibrator Kit (List No. 2N40-70)
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1. Abbott RealTime HBV Calibrator A (List No. 2G34A)
(12 vials, 1.3 mL per vial)
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• Less than 0.01% noninfectious linearized HBV DNA plasmid in a buffer solution.
Preservatives: 0.085% Sodium azide and 0.15% ProClin 950.
or
2. Abbott RealTime HBV Calibrator B (List No. 2G34B)
(12 vials, 1.3 mL per vial)
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• Less than 0.01% noninfectious linearized HBV DNA plasmid in a buffer solution.
Preservatives: 0.085% Sodium azide and 0.15% ProClin 950.
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WARNINGS AND PRECAUTIONS
In Vitro Diagnostic Medical Device
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For In Vitro Diagnostic Use Only.
The Abbott RealTime HBV assay is not intended for use in the screening of blood,
plasma, or tissue donors for HBV, or to be used as a diagnostic test to confirm the
presence of HBV infection.
Use only USP Grade 190-200 Proof Ethanol (95-100% Ethanol) to prepare the
mWash2DNA sample preparation reagent. Do not use ethanol that contains denaturants.
Safety Precautions
Refer to the Hazard Sections of the Abbott m2000sp Operations Manual and the Abbott
m2000rt Operations Manual for instructions on safety precautions.
CAUTION: This preparation contains human sourced and/or potentially infectious
components. Components sourced from human blood have been tested and found to
be nonreactive by FDA-licensed tests for antibody to HCV, antibody to HIV-1, antibody
to HIV-2, and HBsAg. The material is also tested and found to be negative by FDA-
licensed PCR methods for HIV-1 RNA and HCV RNA. No known test method can
offer complete assurance that products derived from human sources or inactivated
microorganisms will not transmit infection. These reagents and human specimens
should be handled as if infectious using safe laboratory procedures, such as those
outlined in Biosafety in Microbiological and Biomedical Laboratories,14 OSHA Standards
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on Bloodborne Pathogens,15 CLSI Document M29-A3,16 and other appropriate
biosafety practices.17 Therefore all human sourced materials should be considered
infectious.
These precautions include, but are not limited to, the following:
• Wear gloves when handling specimens or reagents.
• Do not pipette by mouth.
• Do not eat, drink, smoke, apply cosmetics, or handle contact lenses in areas
where these materials are handled.
• Clean and disinfect spills of specimens by including the use of a tuberculocidal
disinfectant such as 1.0% sodium hypochlorite or other suitable disinfectant.14
• Decontaminate and dispose of all potentially infectious materials in accordance
with local, state, and federal regulations.17
Components of the Abbott RealTime HBV Calibrator Kit (List No. 2N40 70), Abbott
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RealTime HBV Control Kit (List No. 2N40-80), and the Abbott RealTime HBV
Amplification Reagent Kit (List No. 2N40-90) contain the following components:
• 2-Methyl-2H-isothiazol-3-one
• Reaction mass of: 5-chloro-2-methyl-4-isothiazolin-3-one (EC no. 247-500-7) and
2-methyl-2H-isothiazol-3-one (EC no. 220-239-6)(3:1)
• Reaction mass of: 5-chloro-2-methyl-4-isothiazolin-3-one (EC no. 247-500-7) and
2-methyl-4-isothiazolin-3-one (EC no. 220-239-6) (3:1)
• Sodium azide
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The following warnings apply:
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Warning
H317 May cause an allergic skin reaction.
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EUH032 Contact with acids liberates very toxic gas.
P261 Avoid breathing mist / vapours / spray.
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P280 Wear protective gloves / protective
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clothing / eye protection.
P272 Contaminated work clothing should not be allowed out of the
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workplace.
P333+P313 If skin irritation or rash occurs: Get medical advice /
attention.
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P302+P352 IF ON SKIN: Wash with plenty of water.
P362+P364 Take off contaminated clothing and wash it before reuse.
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P501 Dispose of contents / container in accordance with local
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Special Precautions
Handling Precautions
The Abbott RealTime HBV assay is only for use with human serum and plasma
specimens that have been handled and stored in capped tubes as described in the
SPECIMEN COLLECTION, STORAGE, AND TRANSPORT TO THE TEST SITE section.
During preparation of samples, compliance with good laboratory practices is essential
to minimize the risk of cross-contamination between samples, and the inadvertent
introduction of nucleases into samples during and after the extraction procedure. Proper
aseptic technique should always be used when working with DNA.
Amplification reactions such as PCR are sensitive to accidental introduction of product
from previous amplification reactions. False positive results could occur if either the
clinical specimen or the RealTime reagents used in the amplification step become
contaminated by accidental introduction of even a few molecules of amplification
product. Measures to reduce the risk of contamination in the laboratory include
physically separating the activities involved in performing PCR in compliance with good
laboratory practices.
Work Areas
Use two dedicated areas within the laboratory for performing the Abbott RealTime HBV
assay.
• The Sample Preparation Area is dedicated to processing samples (specimens,
Abbott RealTime HBV Controls and Calibrators) and to adding processed
samples, controls, and calibrators to the Abbott 96-Well Optical Reaction Plate.
The Abbott m2000sp combines the Abbott RealTime HBV amplification reagent
components to create the amplification master mix and transfers aliquots of the
master mix to the reaction plate. All reagents used in the Sample Preparation
Area should remain in this dedicated area at all times. Laboratory coats,
pipettes, pipette tips, and vortexers used in the Sample Preparation Area must
remain in this area and not be moved to the Amplification Area. Do not bring
amplification product into the Sample Preparation Area.
• The Amplification Area is dedicated to the amplification and detection of
amplified product. Laboratory coats and equipment used in the Amplification
Area must remain in this area and not be moved to the Sample Preparation
Area.
Components contained within a kit are intended to be used together. Do not mix
components from different kit lots. For example, do not use the negative control from
control kit lot X with the positive controls from control kit lot Y.
Do not use kits or reagents beyond expiration date.
Work areas and instrument platforms must be considered potential sources of
contamination. Change gloves after contact with potential contaminants (specimens,
eluates, and/or amplified product) before handling unopened reagents, negative control,
positive controls, calibrators, or specimens. Refer to the Abbott m2000sp Operations
Manual and the Abbott m2000rt Operations Manual for instrument cleaning procedures.
If the Abbott m2000sp instrument run is aborted, dispose of all commodities and
reagents according to the Abbott m2000sp Operations Manual. If the Abbott m2000sp
master mix addition protocol is aborted, seal the Abbott 96-Well Optical Reaction Plate After centrifugation, remove serum or plasma from cells. Serum or plasma specimens
in a sealable plastic bag and dispose according to the m2000sp Operations Manual, may be stored:
Hazards section, along with the gloves used to handle the plate. • At 15 to 30°C for up to 24 hours
If the Abbott m2000rt instrument run is interrupted or aborted, seal the Abbott 96 Well • At 2 to 8°C for up to 3 days

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Optical Reaction Plate in a sealable plastic bag and dispose according to the Abbott
m2000rt Operations Manual along with the gloves used to handle the plate.
Decontaminate and dispose of all specimens, reagents, and other potentially
biohazardous materials in accordance with local, state, and federal regulations.17
All materials should be handled in a manner that minimizes the chance of potential
contamination of the work area. Note: Autoclaving the sealed Reaction Plate will not
eliminate the amplified product and may contribute to the release of the amplified
product by opening of the seal. The laboratory area can become contaminated with
amplified product if the waste materials are not carefully handled and contained
before and after processing.
Aerosol Containment
To reduce the risk of nucleic acid contamination due to aerosols formed during manual
pipetting, aerosol barrier pipette tips must be used for all manual pipetting. The pipette
tips must be used only one time. Clean and disinfect spills of specimens and reagents
as stated in the Abbott m2000sp or the Abbott m2000rt Operations Manuals.
Contamination and Inhibition
• At -20°C or colder for longer term
Multiple freeze-thaw cycles should be avoided. If frozen, thaw specimens at 15 to 30°C
or at 2 to 8°C. Once thawed, if specimens are not being processed immediately, they
can be stored at 2 to 8°C for up to 6 hours.
Specimen Transport
Ship specimens frozen on dry ice. For domestic and international shipments, specimens
should be packaged and labeled in compliance with applicable state, federal, and
international regulations covering the transport of clinical, diagnostic, or biological
specimens.
INSTRUMENT PROCEDURE
The Abbott RealTime HBV application file(s) must be installed on the Abbott m2000sp
and Abbott m2000rt instruments from the Abbott RealTime HBV m2000 System
Combined Application CD-ROM prior to performing the assay. For detailed information
on application file installation, refer to the Abbott m2000sp and m2000rt Operations
Manuals, Operating Instructions section.

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The following precautions should be observed to minimize the risks of DNase ABBOTT REALTIME HBV ASSAY PROCEDURE

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contamination, cross-contamination between samples, and inhibition: Materials Provided
• Wear appropriate personal protective equipment at all times. • Abbott RealTime HBV Amplification Reagent Kit (List No. 2N40-90)
• Use powder-free gloves.

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• Change gloves after contact with potential contaminants (specimens, eluates, Materials Required But Not Provided
and/or amplified product). • Abbott RealTime HBV Control Kit (List No. 2N40-80)

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• To reduce the risk of nucleic acid contamination due to aerosols formed during • Abbott RealTime HBV Calibrator Kit (List No. 2N40-70)
manual pipetting, pipettes with aerosol barrier tips must be used for all pipetting. Sample Preparation Area

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The length of the tip should be sufficient to prevent contamination of the pipette • Abbott m2000sp
barrel. While pipetting, care should be taken to avoid touching the pipette barrel • Abbott mSample Preparation SystemDNA (4 x 24 Preps) (List No. 06K12-24)

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to the inside of the sample tube or container. The use of extended aerosol • Abbott Proteinase K (List No. 03L78-061)
barrier pipette tips is recommended. • Abbott RealTime HBV m2000 System Combined Application CD-ROM
• Change aerosol barrier pipette tips between ALL manual liquid transfers. (List No. 2N43)

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• Clean and disinfect spills of specimens and reagents as stated in the Abbott • Sample Racks
m2000sp and the Abbott m2000rt Operations Manuals, Hazards section. • 5 mL Reaction Vessels
• Replace any empty or partially used 200 µL and 1000 µL disposable tip trays with • 200 mL Reagent Vessels

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full trays before every run. • Master Mix Vial
• The Abbott mSample Preparation SystemDNA (4 x 24 Preps) and Abbott Proteinase • Abbott 96-Well Optical Reaction Plate
K reagents are single use only. Use new reagent vessels, reaction vessels, and • Abbott 96-Deep Well Plate
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newly opened reagents for every new Abbott RealTime HBV assay run. At the • Abbott Splash-Free Support Base
• Abbott Optical Adhesive Cover
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end of each run, discard all remaining reagents from the worktable as stated in
the Abbott m2000sp Operations Manual and the Abbott mSample Preparation • Abbott Adhesive Cover Applicator
• Round-bottom 12.5 x 75 mm Sample Tubes
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SystemDNA (4 x 24 Preps) and Abbott Proteinase K package inserts.

• Vortex Mixer
STORAGE INSTRUCTIONS • 50 mL Polypropylene Centrifuge Tubes
• Centrifuge capable of 2,000g
Abbott RealTime HBV Amplification Reagent Kit (List No. 2N40-90) • Calibrated Precision Pipettes capable of delivering 10 µL-1000 µL
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• 20 µL-1000 µL Aerosol Barrier Pipette Tips for precision pipettes

The Abbott RealTime HBV Amplification Reagent Pack and Internal
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• Serological Pipettes
Control vials must be stored at -10°C or colder when not in use. Care
• Graduated Cylinder, 100 mL
must be taken to separate the Abbott RealTime HBV Amplification
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• USP Grade 190-200 Proof Ethanol (95-100% Ethanol). Do not use ethanol that
Reagent Pack that is in use from direct contact with samples,
calibrators, and controls. contains denaturants.
• Molecular Biology Grade Water
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Abbott RealTime HBV Control Kit (List No. 2N40-80) • 1.7 mL Molecular Biology Grade Microcentrifuge Tubes (Dot Scientific, Inc. or
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equivalent)*
The Abbott RealTime HBV Negative and Positive Controls must be • Cotton Tip Applicators (Puritan or Equivalent)*
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stored at -10°C or colder.

*Note: These items are used in the procedure for Monitoring the Laboratory for the
Presence of Amplification Product. Refer to the QUALITY CONTROL PROCEDURES
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Abbott RealTime HBV Calibrator Kit (List No. 2N40-70) section of this package insert.
Amplification Area
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The Abbott RealTime HBV Calibrator A and Calibrator B must be

• Abbott m2000rt
stored at -10°C or colder.
• Abbott RealTime HBV m2000 System Combined Application CD-ROM
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(List No. 2N43)

SHIPPING CONDITIONS • Abbott m2000rt Optical Calibration Kit (List No. 4J71-93)
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• Abbott RealTime HBV Amplification Reagent Kit: Ship on dry ice. Other Materials
• Abbott RealTime HBV Control Kit: Ship on dry ice. • Biological safety cabinet approved for working with infectious materials.
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• Abbott RealTime HBV Calibrator Kit: Ship on dry ice.
INDICATION OF INSTABILITY OR DETERIORATION OF
REAGENTS
When a positive or negative control value is out of the expected range, it may indicate
deterioration of the reagents. Associated test results are invalid and samples must
be retested. Assay recalibration may be necessary. Refer to the QUALITY CONTROL
PROCEDURES: Assay Calibration section of this package insert for details.
If you receive reagents, calibrators, or controls that are in a condition contrary to label
recommendation, or that are damaged, contact Abbott Molecular Technical Services.
SPECIMEN COLLECTION, STORAGE, AND TRANSPORT TO
THE TEST SITE
Specimen Collection and Storage
Human serum and plasma (EDTA) specimens may be used with the Abbott RealTime
HBV assay. Follow the manufacturer’s instructions for processing collection tubes.
Freshly drawn specimens (whole blood) may be held at 2 to 30°C for up to 6 hours
prior to centrifugation.
• Sealable plastic bags
Procedural Precautions
Read the instructions in this package insert carefully before processing samples.
The Abbott RealTime HBV Calibrators, Internal Control, Negative Control, and Low and
High Positive Control vials are intended for single-use only and should be discarded
after use.
Sample tubes should be inspected for air bubbles. If found, remove them with a sterile
pipette tip. Reagent bubbles may interfere with proper detection of reagent levels in
the reagent vessel, causing insufficient reagent aspiration, which could impact results.
Caution should be taken to avoid cross-contamination between samples by using a
new sterile pipette tip for each tube.
Use aerosol barrier pipette tips or disposable pipettes only one time when pipetting
specimens, controls, calibrators, or Amplification Reagents. To prevent contamination
to the pipette barrel while pipetting, care should be taken to avoid touching the pipette
barrel to the inside of the sample tube or container. The use of extended aerosol
barrier pipette tips is recommended.
Monitoring procedures for the presence of amplification product can be found in the

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QUALITY CONTROL PROCEDURES section in this package insert.
To reduce the risk of nucleic acid contamination, clean and disinfect spills of
specimens by including the use of a tuberculocidal disinfectant such as 1.0% sodium
hypochlorite or other suitable disinfectant.
A calibration curve must be established before specimens are tested. The use of the
Abbott RealTime HBV Calibrators and Controls is integral to the performance of the
Abbott RealTime HBV assay. Refer to the QUALITY CONTROL PROCEDURES section
of this package insert for details.
ASSAY PROTOCOL
Sample Preparation Area
All specimen storage and preparation must take place in the dedicated Sample
Preparation Area. Refer to the Handling Precautions section of this package insert for
instructions before preparing samples.
For a detailed description of how to operate the Abbott m2000sp instrument and Abbott
m2000rt instrument, refer to the Abbott m2000sp and m2000rt Operations Manuals,
Operating Instructions section.
Laboratory personnel must be trained to operate the Abbott m2000sp and m2000rt
instruments. The operator must have a thorough knowledge of the applications run on
the instruments and must follow good laboratory practices.
1. A maximum of 48 samples can be processed in each run. A negative control, a low
positive control, and a high positive control must be included in each run, therefore
allowing a maximum of 45 specimens to be processed per run.
NOTE: If performing a run of more than 24 samples, empty the solid waste
container before the run and replace with a new biohazard bag if any waste
is present.
• Check sample volume. The Abbott RealTime HBV assay minimum sample volume
and associated rack requirements on the Abbott m2000sp are:
Abbott RealTime HBV
Minimum Sample Volume
Assay Application
Rack Tube Diameter* 0.2 mL 0.5 mL
13 mm 11.5 mm - 14.0 mm 0.4 mL - 0.8 mL 0.7 mL - 1.2 mL
16 mm 14.5 mm - 16.0 mm 0.4 mL - 1.0 mL 0.8 mL - 1.4 mL
* Refers to the sample tube outer diameter.
• Minimum sample volume varies with tube geometry and size. Refer to the
m2000sp Operations Manual and QUICK REFERENCE GUIDE FOR SAMPLE
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TUBE SIZES AND VOLUMES for recommended sample input volume.
• If frozen, thaw specimens at 15 to 30°C or at 2 to 8°C. Once thawed, if specimens
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are not being processed immediately, store at 2 to 8°C for up to 6 hours.
• Before use, vortex specimens three times for 2 to 3 seconds. Ensure that
bubbles or foam are not created. If found, remove them with a new sterile
pipette tip for each tube. Specimens showing particulate matter or turbidity
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should be clarified by centrifugation at 2,000g for 5 minutes prior to testing.
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Aliquot each specimen into clean tubes or vials if necessary. Refer to the Abbott
m2000sp Operations Manual for tube sizes. Avoid touching the inside of the cap
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when opening tubes.
2. Thaw assay controls and Internal Control (IC) at 15 to 30°C or at 2 to 8°C. Thaw
calibrators at 15 to 30°C or at 2 to 8°C only if performing a calibration run; see
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QUALITY CONTROL PROCEDURES section of this package insert.
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• Once thawed, if calibrators, controls, and IC are not being processed immediately,
store at 2 to 8°C for up to 24 hours.
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• Vortex each assay calibrator and each control three times for 2 to 3 seconds
before use. Ensure that bubbles or foaming are not created. If found, remove
m
them with a new sterile pipette tip for each tube. Ensure that the contents of the
vials are at the bottom after vortexing by tapping the vials on the bench to bring
or
liquid to the bottom of the vials.
3. Thaw amplification reagents at 15 to 30°C or at 2 to 8°C until required for the
nf
amplification master mix procedure. This step can be initiated before completion of
the sample preparation procedure.
rI
Note: Do not vortex the Amplification Reagent Pack.
• Once thawed, the amplification reagents can be stored at 2 to 8°C for up to 24
Fo
hours if not used immediately.
4. Open the Abbott Proteinase K reagent pack. Add 17.15 mL of Molecular Biology
Grade water to a 50 mL polypropylene centrifuge tube. Pipet 2.45 mL of Proteinase
K into the container of water. Mix by gentle inversion 10 to 15 times. Transfer the
entire contents to a reagent vessel labeled with the Proteinase K barcode label.
Place the reagent vessel in reagent carrier #1 location 2.
NOTE: The following steps 5 through 9 pertain to the use of the mSample
Preparation SystemDNA kit (List No. 06K12-24).
NOTE: Use one bottle of Proteinase K solution, one set of the mSample
Preparation SystemDNA reagents, one vial of IC, and one RealTime HBV
Amplification Reagent Pack to support up to 24 reactions. Use a second
set of reagents to support 25 to 48 reactions, with the exception of the
mMicroparticlesDNA. One bottle of mMicroparticlesDNA will support up to 48
reactions. Do not use more than one bottle of mMicroparticlesDNA.
5. Open the Abbott mSample Preparation pack. If crystals are observed in any of the
reagent bottles upon opening, allow the reagents to equilibrate at room temperature
4
until the crystals disappear. Do not use the reagents until the crystals have
dissolved.
6. Prepare the mWash2DNA by adding 70 mL of USP Grade 190-200 Proof Ethanol
(95-100% Ethanol) to the mWash2DNA bottle as described in the Abbott mSample
Preparation SystemDNA product information. Do not use ethanol that contains
denaturants.
7. Vortex the IC vial(s) three times for 2 to 3 seconds before use.
8. Using a calibrated precision PIPETTE DEDICATED FOR INTERNAL CONTROL USE
ONLY, add 100 µL of IC to a bottle of mLysis Buffer. Mix by gently inverting the
container 5 to 10 times to minimize foaming.
9. Gently invert the Abbott mSample Preparation bottles to ensure a homogeneous
solution and pour the contents into the appropriate reagent vessels per the Abbott
m2000sp Operations Manual, Operating Instructions.
10. Place the low and high positive controls, the negative control, the calibrators (if
applicable), and the patient specimens into the m2000sp sample rack.
11. Place the 5 mL Reaction Vessels into the m2000sp 1 mL subsystem carrier.
12. Load the carrier racks containing the Abbott mSample Preparation reagents and
Proteinase K, and the Abbott 96-Deep Well Plate, on the Abbott m2000sp worktable
as described in the Abbott m2000sp Operations Manual, Operating Instructions.
13. From the Run Sample Extraction screen, select the appropriate application file
corresponding to the sample volume being tested. Initiate the sample extraction
y
protocol as described in the m2000sp Operations Manual, Operating Instructions.
op
• Enter calibrator (needed if a calibration curve has not been stored on the
m2000rt) and control lot specific values in the Sample Extraction: Assay Details
screen. Lot specific values are specified in each Abbott RealTime HBV Calibrator
C
and Control Kit card.
d
NOTE: Verify the values entered match the values on the kit cards.
lle
• The Abbott m2000sp Master Mix Addition protocol (step 14) must be initiated
within 60 minutes after completion of the Sample Extraction protocol.
tro
NOTE: Change gloves before handling the amplification reagents.
14. Load the amplification reagents, the master mix vial, and the 96-well Optical
on
Reaction Plate on the m2000sp worktable after sample preparation is completed.
• Each Amplification Reagent Pack supports up to 24 reactions.
• Ensure the amplification reagents are thoroughly thawed before use.
C
• Prior to opening the amplification reagents, ensure that the contents are at the
bottom of the vials by tapping the vials in an upright position on the bench.
a • Remove and discard the amplification vial caps.
• A second Amplification Reagent Pack is required if performing 25 to 48 reactions.
15. Select the appropriate deep well plate from the Run Master Mix Addition screen
that matches the corresponding sample preparation extraction. Initiate the Abbott
m2000sp Master Mix Addition protocol. Follow the instructions as described in the
Abbott m2000sp Operations Manual, Operating Instructions section.
• The m2000rt protocol (step 20) must be started within 60 minutes of the
completion of the Master Mix Addition protocol (step 15).
Amplification Area
16. Switch on and initialize the Abbott m2000rt in the Amplification Area.
• The Abbott m2000rt requires 15 minutes to warm up.
NOTE: Remove gloves before returning to the Sample Preparation Area.
Sample Preparation Area
17. Seal the Abbott 96-Well Optical Reaction Plate after the Abbott m2000sp
instrument has completed addition of samples and master mix according to the
Abbott m2000sp Operations Manual, Operating Instructions section.
NOTE: Since a maximum of 48 samples can be processed in each run, the
96-Well Optical Reaction Plate will contain empty wells.
18. Place the 96-Well Optical Reaction Plate into the Splash-Free Support Base for
transfer to the Abbott m2000rt instrument.
19. Export the completed 96-Well Optical Reaction Plate results to a CD or Network
Drive.
Amplification Area
20. Place the Abbott 96-Well Optical Reaction Plate in the Abbott m2000rt instrument.
Import the m2000sp test order via CD or Network Drive per the Import Order
instructions in the Abbott m2000rt Operations Manual, Operating Instructions
section.
POST PROCESSING PROCEDURES
21. Remove the Abbott 96-Deep Well Plate from the worktable and dispose according
to the Abbott m2000sp Operations Manual.
22. Place the Abbott 96-Well Optical Reaction Plate in a sealable plastic bag and
dispose according to the Abbott m2000rt Operations Manual, along with the gloves
used to handle the plate.
23. Clean the Splash Free Support Base before next use, according to the Abbott
m2000rt Operations Manual.
QUALITY CONTROL PROCEDURES
Abbott m2000rt Optical Calibration
Refer to the Calibration Procedures section in the Abbott m2000rt Operations Manual
for a detailed description of how to perform an Abbott m2000rt Optical Calibration.
Optical calibration of the Abbott m2000rt instrument is required for the accurate
measurement and discrimination of dye fluorescence during the Abbott RealTime HBV 6. Add 20 µL of the mWash 1 buffer to each microcentrifuge tube.

assay. 7. Cap the microcentrifuge tube.

The following Abbott m2000rt Optical Calibration Plates are used to calibrate the Abbott 8. Test this sample according to the assay procedure section of this package insert.

m2000rt instrument for the Abbott RealTime HBV assay: 9. Transfer liquid from microcentrifuge tube to a 5 mL Reaction Vessel.

• FAM™ Plate (Carboxyfluorescein) 10. Bring the volume to 1.5 mL with Molecular Biology Grade water.

• ROX™ Plate (Carboxy-X-rhodamine) 11. The presence of contamination is indicated by the detection of HBV in the swab

• VIC® Plate (Proprietary dye) samples.
Assay Calibration 12. If HBV is detected on equipment, follow the cleaning and decontaminating

A calibration curve is required to quantitate HBV DNA in the specimens and controls.
Two assay calibrators are run in replicates of three to generate a calibration curve
(HBV concentration [log IU/mL] versus the threshold cycle [Ct] at which a reactive level
of fluorescent signal is detected). The lot specific values for Calibrator A and Calibrator
B are specified on each Abbott RealTime HBV Calibrator Kit Card and must be entered
into the assay test order when a run is performed. The calibration curve slope and
guidelines given in that equipment’s operations manual. If HBV is detected on
surfaces, clean the contaminated areas with 1.0% (v/v) sodium hypochlorite
solution, followed by 70% ethanol or water. Note: Chlorine solutions may pit
equipment and metal. Use sufficient amounts or repeated applications of 70%
ethanol or water until chlorine residue is no longer visible.
13. Repeat testing of the contaminated area by following Steps 1 through 10.

intercept are calculated and stored on the instrument. The concentration of HBV DNA in
a sample is calculated from the calibration curve. Results are automatically reported on
the m2000rt workstation.
The Low and High Positive Controls and Negative Control must be included in the
calibration run.
Follow the procedure for sample extraction, reagent addition, amplification and detection
RESULTS
Calculation
The concentration of HBV DNA in a sample or control is calculated from either a stored
calibration curve, or a calibration curve created by calibrators within a calibration or
sample run. The Abbott m2000rt instrument automatically reports the results on the

y
protocols as stated in the Abbott m2000sp Operations Manual and the m2000rt m2000rt workstation. Assay results are reported in IU/mL or log IU/mL. Results can also
be reported in copies/mL or log copies/mL using a conversion factor of 3.41

op
Operations Manual. Ensure that assay control values observed in the final report are
within the ranges specified on the Abbott RealTime HBV Control Kit Card. (1 IU = 3.41 copies). Note: The assay is calibrated to the WHO International HBV
Once an Abbott RealTime HBV calibration is accepted and stored, it may be used DNA Standard. The 3.41 conversion factor is based on an average conversion factor

C
for 6 months. During this time, all subsequent samples may be tested without further across the assay dynamic range.
calibration unless: The following table represents the potential m2000rt outputs that can be observed by
the user.

d
• An Abbott RealTime HBV Amplification Reagent Kit with a new lot number is used.
• An Abbott mSample Preparation SystemDNA (4 x 24 Preps) with a new lot number Interpretation of Results

lle
is used.
• An Abbott RealTime HBV application specification file for a different sample Sample
Volume Result Interpretation

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volume is used.
• An updated version of the Abbott RealTime HBV application specification file is 0.5 mL Not Detected Target not detected
installed. < 1.00 Log IU/mLa Detectedc

on
Detection of Inhibition 1.00 to 9.00 Log IU/mL d

An IC threshold cycle [Ct] assay validity parameter is established during a calibration run. > 9.00 Log IU/mL > ULQe

C
Prior to sample preparation, a defined, consistent quantity of the IC is introduced into
the lysis buffer, which is then used during the processing of each specimen, calibrator, 0.2 mL Not Detected Target not detected
and control, and measured on the m2000rt instrument to demonstrate proper sample a < 1.18 Log IU/mLb Detectedc
processing and assay validity. The IC is composed of a DNA sequence unrelated to the 1.18 to 9.00 Log IU/mL d
HBV DNA sequence.
ot
> 9.00 Log IU/mL > ULQe
The median amplification cycle at which the IC target sequence fluorescent signal is
detected in calibration samples establishes the IC Ct validity range to be met by all a
10 IU/mL
-N

subsequent processed specimens using that calibration curve. b
15 IU/mL

An error is displayed when a specimen or control fails to meet this specification. Refer
c
Below LLQ (lower limit of quantitation or LLoQ); HBV DNA is not quantifiable.

d
Calculated results are within assay linear range. If a calculated result is obtained, the
to the Abbott m2000rt Operations Manual for an explanation of the corrective actions


Interpretation field is left blank.
for the error code. Specimens whose IC Ct value falls outside of the established range

y

e
> ULQ – above upper limit of quantitation or ULoQ; if IU/mL results are above the linear
must be retested starting with sample preparation.

range of the assay, results are reported as “>1,000,000,000 IU/mL HBV DNA.”
nl

Negative and Positive Controls

If negative or positive controls are out of range, all of the specimens and controls from
O

A negative control, a low positive control, and a high positive control are included in
each run to evaluate run validity. that run must be reprocessed, beginning with sample preparation.
The lot specific values for the low positive control and high positive control are specified If quantitative results are desired for those specimens reported as > ULQ (ULoQ), the
n

on each Abbott RealTime HBV Control Kit Card and must be entered into the test order original specimen should be diluted 1:50 with HBV-negative human plasma or serum
(consistent with the matrix of the original specimen), and the test repeated. Multiply
io

when a run is performed.

the reported result by the dilution factor of 50 to obtain the quantitative result. Example
An error is displayed when a control result is out of range. Refer to the Abbott m2000rt calculations are provided below.
at

Operations Manual for an explanation of the corrective actions for the error code. If
negative or positive controls are out of range, all of the specimens and controls from Calculate 10x Multiply whole number
m

that run must be reprocessed, beginning with sample preparation. (x = result in log result by 50 (dilution Calculate log (y)
The presence of HBV must not be detected in the negative control. HBV detected in Example result for unit); round to whole factor) to obtain result (y = whole number
or

diluted specimen number corrected for dilution result)

the negative control is indicative of contamination by other samples or by amplified
product introduced during sample preparation or during preparation of the Abbott 96- 7.59 log IU/mL 38,904,515 IU/mL 1,945,225,750 IU/mL 9.29 log IU/mL
nf

Well Optical Reaction Plate. To avoid contamination, clean the Abbott m2000sp and 63,095,734 IU/mL Not applicable 3,154,786,700 IU/mL Not applicable
m2000rt instruments and repeat the sample processing for controls and specimens
rI

following the Procedural Precautions. If negative controls are persistently reactive, 8.80 log copies/mL 630,957,345 31,547,867,250 10.50 log copies/mL
contact your Area Customer Support representative. copies/mL copies/mL
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Monitoring the Laboratory for the Presence of Amplification Product
It is recommended that this test be done at least once a month to monitor laboratory
surfaces and equipment for contamination by amplification product. It is very important
to test all areas that may have been exposed to processed specimens and controls,
calibrators, and/or amplification product. This includes routinely handled objects such
as pipettes, the Abbott m2000sp and m2000rt function keys, laboratory bench surfaces,
microcentrifuges, and centrifuge adaptors.
1. Add 0.8 mL Molecular Biology Grade water to a 1.7 mL DNase-free microcentrifuge
tube.
2. Saturate the cotton tip of an applicator (Puritan or equivalent) in the Molecular
Biology Grade water from the microcentrifuge tube.
3. Using the saturated cotton tip of the applicator, wipe the area to be monitored
using a sweeping motion. Place the applicator into the microcentrifuge tube.
4. Swirl the cotton tip in Molecular Biology Grade water 10 times, and then press
the applicator along the inside of the tube so that the liquid drains back into the
solution at the bottom of the microcentrifuge tube. Discard the applicator.
5. Pipette 0.5 mL of the mWash 1 buffer to a clean tube using the pipette dedicated
for Internal Control use.
49,905,245 Not applicable 2,495,262,250 Not applicable
copies/mL copies/mL
LIMITATIONS OF THE PROCEDURE
• FOR IN VITRO DIAGNOSTIC USE ONLY.
• Optimal performance of this test requires appropriate specimen collection,
storage, and transport to the test site (refer to the SPECIMEN COLLECTION,
STORAGE, AND TRANSPORT TO THE TEST SITE section of this package insert).
• Human serum and plasma (EDTA) may be used with the Abbott RealTime HBV
assay. The use of other anticoagulants has not been validated for use with the
Abbott RealTime HBV assay.
• The use of specimens collected in serum tubes that contain Z-clot activator, or
similar types of rapid clot activator, may cause inhibited results in the RealTime
HBV assay. Therefore, serum collection tubes containing Z-clot activator or
similar rapid clot activators should not be used.
• The instruments and assay procedures reduce the risk of contamination by
amplification product. However, nucleic acid contamination from the calibrators,
positive controls, or specimens must be controlled by good laboratory practice

5
 and careful adherence to the procedures specified in this package insert.
• In rare cases, a very low level positive result may occur from cross contamination
during processing of an extremely high copy number adjacent specimen.
Carryover rates in representative studies ranged from 0% to 2%. Per treatment
guidelines, a 1 log increase is needed in order to impact patient management.18,19
In addition, treatment guidelines require two consecutive elevated measurements
to occur before changing patient management.20
• A single Entecavir mutation (rtA97V) occurs within the reverse primer. Of all
known resistance mutations, it is the only one that occurs within any Abbott
RealTime HBV primer or probe sequence. Software simulation predicts that this
mutation (rtA97V) would not be expected to interfere with assay results when
using RealTime HBV assay conditions.
• A specimen with a result of “Not Detected” cannot be presumed to be negative
for HBV DNA.
• Precision was established with HBV Genotypes A and C only.
• Drug interference was evaluated using a plasma matrix, and was not evaluated in
serum. The listed drugs were tested in pools, and individual drug effects were not
assessed.
• The interference studies were performed with an HBV DNA concentration of
2,933 IU/mL (3.47 log IU/mL). Potential interference on HBV DNA concentrations
close to the assay LLQ (LLoQ) was not assessed.
targeted from 9.13 log IU/mL to 0.29 log IU/mL in HBV negative human plasma was
tested and evaluated in accordance with methods defined in the CLSI EP6-A,21 using
the 0.5 mL sample preparation protocol. The Abbott RealTime HBV assay was shown
to be linear in plasma across the range of HBV DNA concentrations tested (shown in
Figure 2) with deviation from linearity of not more than 0.20 log IU/mL.
In a second study, one panel consisting of Genotype A and one panel consisting of
Genotype C were tested. The two 10-member panels were prepared by diluting to
concentrations targeted from 1.27 log IU/mL to 8.47 log IU/mL for Genotype A and
1.59 log IU/mL to 8.79 log IU/mL for Genotype C. The two panels were prepared
with high copy HBV-positive specimens diluted in HBV serologically-negative human
plasma. Least squares linear regression analysis was performed for Genotypes A and C
separately. Analysis for Genotype A is shown in Figure 3 and analysis for Genotype C is
shown in Figure 4. The Abbott RealTime HBV assay was shown to be linear in plasma
across the range of HBV DNA concentrations tested for HBV Genotype A and HBV
Genotype C.
Figure 2
Abbott RealTime HBV Linear Range
Least Squares Linear Regression Analysis

y
• Some of the cross-reactivity studies were performed with nucleic acids (DNA y = 0.99x + 0.01
and RNA) only. For further detail, refer to the SPECIFIC PERFORMANCE

op
r = 0.999
n = 77
CHARACTERISTICS section of this package insert.
• Results from the Abbott RealTime HBV assay should be interpreted in conjunction

C
with other clinical and laboratory findings.

Abbott Realtime HBV (Log IU/mL)

SPECIFIC PERFORMANCE CHARACTERISTICS

d
The following performance characteristics were determined using the RealTime HBV

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assay with the 0.5 mL sample preparation procedure, unless otherwise specified.
WHO STANDARDIZATION

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Figure 1 demonstrates the comparison of Abbott RealTime HBV Assay Calibrators to
the WHO International HBV DNA Standard. Abbott RealTime HBV Calibrators trace to
the World Health Organization (WHO) International Standard for Hepatitis B Virus DNA

on
(NIBSC) each time a lot is manufactured. Each lot of calibrator is specifically assigned
a quantitation value through testing with HBV Primary Calibrators, which are directly
tested against the WHO standard. The lot-specific quantitation values for each HBV

C
calibrator are entered into the m2000rt software when a run is being performed.
The evaluation was conducted with the WHO 1st International HBV DNA Standard, and a
one lot of HBV Calibrators, and was performed on one run. The WHO standard was
reconstituted to a concentration of 1 x 105 IU/mL and then diluted to 1 x 104, 1 x 103,
ot
and 1 x 102 IU/mL in negative human plasma. The highest assay calibrator, Calibrator
B, which is lot-assigned at 6.42 log IU/mL, was diluted to 1 x 105,1 x 104, 1 x 103, and
Target Concentration (Log IU/mL)
1 x 102 IU/mL in Tris-EDTA (TE) buffer. The data for Calibrator B and its dilution series
-N

are presented in comparison to the WHO standard dilution series in Figure 1. The
results indicate that the assay standardization process provides quantitation values for Figure 3
the RealTime HBV Calibrators and the WHO standard that are similar to the expected Abbott RealTime HBV Linear Range
values, with deviation of not more than 0.33 log IU/mL. The maximum deviation was Least Squares Linear Regression Analysis
y

obtained at the assay ULQ (ULoQ). Genotype A

nl

9
O

Figure 1 y = 0.99x - 0.10

r = 0.998
Comparison of WHO 1st International HBV DNA Standard 8 n = 80
with Abbott RealTime HBV Calibrators
n

7
io

Abbott Realtime HBV (Log IU/mL)

at

6
m

5
or

4
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3
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2
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0
0 1 2 3 4 5 6 7 8 9

Target Concentration (Log IU/mL)

LINEAR RANGE
The ULQ (ULoQ) for the Abbott RealTime HBV assay is 109 IU/mL (9.00 log IU/mL)
and the LLQ (LLoQ) is equivalent to the LoD, which is 10 IU/mL (1.00 log IU/mL) for
the 0.5 mL sample preparation protocol and 15 IU/mL (1.18 log IU/mL) for the 0.2 mL
 
 
sample preparation protocol.
In one study, a 13-member panel prepared by diluting an HBV-positive specimen

6
 Figure 4 LINEARITY OF ASSAY BY HBV GENOTYPES
The ability of the RealTime HBV assay to detect and quantitate HBV genotypes was
Abbott RealTime HBV Linear Range evaluated through linearity studies by diluting eight specimens, one of each genotype A
Least Squares Linear Regression Analysis through H, to target concentrations of 4.47 log IU/mL, 3.47 log IU/mL, 2.47 log IU/mL,
Genotype C

 
and 1.17 log IU/mL in HBV serologically negative human plasma. Three replicates were
9 tested at each concentration for each genotype, using the 0.5 mL sample preparation
y = 0.99x - 0.09 protocol. Data of the studies demonstrated that RealTime HBV assay is capable to
r = 0.999
8 n = 80
quantitate different HBV genotypes across linear range with deviation of not more than
0.51 log IU/mL. The results are summarized in Table 3 and Figure 5.
7
Table 3
Abbott Realtime HBV (Log IU/mL)

6 Abbott RealTime HBV

Linearity of Assay by HBV Genotypes
5
Maximum Differencea
Between Genotype A and
4 Linear Equation from Corresponding Genotype
Genotype Linearity Study (Log IU/mL)
3 A y = 0.95x + 0.19 n/a
B y = 0.89x + 0.40 0.35

y
2 C y = 0.93x + 0.32 0.11

op
D y = 0.89x + 0.43 0.32
1 E y = 0.86x + 0.58 0.44
F y = 0.90x + 0.42 0.23

C
0
G y = 0.85x + 0.61 0.51
0 1 2 3 4 5 6 7 8 9
H y = 0.86x + 0.60 0.42

d
Target Concentration (Log IU/mL)
a
The maximum difference was obtained at the assay ULQ (ULoQ) or LLQ (LLoQ).

lle
Limit of Quantitation Figure 5

tro
The total analytical error (TAE) was calculated using estimates determined from the Abbott RealTime HBV
reproducibility studies that were conducted at three sites: two external sites and one Linearity of Assay by HBV Genotypes
internal site. Genotypes A and C were tested at both sample volumes and in both (Dilutional Linearity)

on
plasma and serum.
Presented in Table 1 are the TAE estimates for the plasma panel members that had an
observed concentration at or near the assay limit of detection, for each sample input

C
volume. Presented in Table 2 are the TAE estimates for the serum panel study. TAE was
estimated by two different methods (see table footnotes). a
These studies demonstrated that the Abbott RealTime HBV assay can determine with
an acceptable level of accuracy the concentration of HBV DNA in EDTA plasma and
ot
serum at concentrations of 10 IU/mL (1.00 log IU/mL) for the 0.5 mL sample protocol
volume and 15 IU/mL (1.18 log IU/mL) for the 0.2 mL sample protocol volume. At these
-N

concentrations, the difference between two measurements of more than 1.00 log IU/mL
is statistically significant.

Table 1
y

Abbott RealTime HBV

nl

Total Analytical Error (TAE) Estimates (Plasma)

(Log IU/mL)
O

HBV TAEc
Sample Genotype Absolute TAEd
n

Volume (Panel Expected Observed Absolute Bias + SQRT (2)

io

(mL) n Member) Conc. Conc. Bias SDb (2 x SD) x 2 x SD

0.5 110 A (5) 1.04 0.90a 0.14 0.32 0.78a 0.91a
at

0.5 119 C (10) 1.14 1.13 0.01 0.29 0.59 0.82

0.2 37 A (5) 1.04 1.03a 0.01 0.40 0.81a 1.13a
m

0.2 46 C (10) 1.14 1.24 0.10 0.30 0.70 0.85

a
Panel Member is below the assay LoD (1.00 log IU/mL for 0.5 mL and 1.18 log IU/mL for 0.2 mL).
or

TAE is provided for information only.

b
SD = Within-run component variability + Between-run component variability.
nf

c
Per section 5.1 of EP17-A CLSI guideline.22
d
Based on difference between two measurements approach.
rI

Table 2
Abbott RealTime HBV
Fo

Total Analytical Error (TAE) Estimates (Serum) WITHIN-LABORATORY PRECISION: LOT-TO-LOT

(Log IU/mL) The precision of the assay was evaluated using an eight-member panel. Panel
members 2, 3, 5, and 7 were prepared by diluting a high copy HBV patient sample in
HBV TAE c
HBV serologically negative human serum. Panel members 1, 4, 6, and 8 were prepared
TAEd
Sample Genotype Absolute by diluting the same high copy HBV patient sample into HBV serologically negative
SQRT (2)
Volume (Panel Expected Observed Absolute Bias + human plasma. A total of three reagent lots were used and each lot was assigned
x 2 x SD
(mL) n Member) Conc. Conc. Bias SDb (2 x SD) an m2000sp and m2000rt instrument pair. A total of 45 replicates were tested for
0.5 88 A (6) 1.36 1.04 0.32 0.20 0.72 0.57 each panel member across the three pairs of m2000sp and m2000rt instruments.
0.5 90 C (12) 1.48 1.29 0.19 0.20 0.59 0.57 One run was performed per day on each instrument pair for five days for a total of 15
0.5 88 C (13) 1.27 0.95a 0.32 0.25 0.82a 0.71a
0.2 88 A (5) 1.56 1.10a 0.46 0.24 0.94a 0.68a runs. Panel members 1 through 8 were run in replicates of three. The concentration
0.2 89 C (12) 1.48 1.14 0.34 0.24 0.82 0.68 levels targeted for the precision panels spanned the linear quantitation range of
a
Panel Member is below the assay LoD (1.00 log IU/mL for 0.5 mL and 1.18 log IU/mL for 0.2 mL). the assay. The 0.5 mL sample preparation protocol was used. The between-lot/
instrument component of precision was less or equal to 0.14 log IU/mL. The results are

TAE is provided for information only.
summarized in Table 4.

b
SD = Within-run component variability + Between-run component variability.
c
Per section 5.1 of EP17-A CLSI guideline.22
d
Based on difference between two measurements approach.

7
 Table 4 Each technician completed one run per day for seven days, for a total of 21 runs. Four
Abbott RealTime HBV replicates were run for each panel member. The SD for between-technician component
Within-Laboratory Precision for the 0.5 mL Sample Preparation Protocol and total SD for the Abbott RealTime HBV assay was found to be less than or equal to
0.06 log IU/mL and 0.11 log IU/mL, respectively, for all panel members greater than the
Between-Lot/ assay limit of detection (1.00 log IU/mL). The results are summarized in Table 5.
Within-Run Between-Run Instrument
Specimen Mean Conc. Component Component Component Total
Panel Typea n (Log IU/mL) SDb SDb SDb SDb,c Table 5
1 P 45 1.41 0.19 0.00 0.08 0.21 Abbott RealTime HBV
2 S 45 2.32 0.07 0.04 0.07 0.11 Within-Laboratory Precision (Operator-to-Operator)
3 S 45 3.48 0.06 0.05 0.08 0.11
Between- Between-
4 P 45 4.38 0.06 0.06 0.08 0.12 Mean Within-Run Run/Day Technician
5 S 45 5.47 0.09 0.00 0.10 0.13 Panel Concentration Component Component Component Total
6 P 45 6.38 0.06 0.07 0.11 0.15 Member n (Log IU/mL) SDa SDa SDa SDa,d
7 S 45 7.54 0.05 0.08 0.14 0.17
8 P 45 8.44 0.04 0.05 0.13 0.14 1 84 8.87 0.06 0.03 0.05 0.08
a
P = Plasma; S = Serum 2 84 6.77 0.05 0.03 0.01 0.06
b
Standard deviations (SD) are in log IU/mL.
c
Total precision includes within-run, between-run and between-lot/instrument components of 3 84 4.53 0.10 0.00 0.05 0.11

precision. 4 84 2.72 0.06 0.02 0.02 0.07
5 73b,c 0.49 0.24 0.00 0.07 0.25

y
WITHIN-LABORATORY PRECISION: OPERATOR-TO-OPERATOR

op
The within-run, between-run, and between-technician (operator) precision of the 6 84 8.57 0.08 0.02 0.06 0.10
Abbott RealTime HBV assay was evaluated by testing 84 replicates of each HBV panel 7 84 6.72 0.07 0.00 0.04 0.08
member that span the dynamic range of the assay from approximately 1.00 log IU/mL to

C
approximately 9.00 log IU/mL, for HBV Genotypes A and C. Panel members 1 through 8 84 4.66 0.09 0.03 0.04 0.10
5 were HBV Genotype A, and panel members 6 through 10 were HBV Genotype C. 9 83c 2.69 0.07 0.03 0.05 0.09

d
The 0.5 mL sample preparation protocol was used for this study. This same panel was
also used as a part of the site-to-site reproducibility study. One lot of amplification 10 84 0.78 0.19 0.00 0.04 0.19

lle
reagents was run on one m2000sp and m2000rt instrument pair by three technicians.
a
Standard Deviations (SD) are in log IU/mL.
b
Target not detected for 10 samples.
c
Error code “Internal Control Failed” for one sample.

tro
d
Total precision includes within-run, between-run/day, and between-technician components of precision.
REPRODUCIBILITY IN PLASMA
The plasma reproducibility panel was tested at three different sites by one technologist and one instrument pair at each site. Panels tested at each site consisted of a 40-member

on
panel (10 unique panel members) that included five concentration levels of one prevalent HBV genotype and five concentration levels of a second prevalent HBV genotype,
repeated four times within the panel. The concentration levels targeted for the reproducibility panels spanned the linear quantitation range of the assay. The HBV genotypes

C
selected for the reproducibility panels were genotype A and genotype C, recognized as prevalent in the U.S. population. Each five-member panel was prepared from a high
copy source sample, which was composed of at least two individual patient specimens that had a common genotype. A total of three reagent lots were used. For the 0.5 mL
reproducibility, each of the three clinical sites tested two of the three lots for five days each. Site 1 used lots A and B, Site 2 used lots B and C, and Site 3 used lots A and C. The
a
0.2 mL reproducibility was tested at each of the three clinical sites using two lots for two days each. The SD for the between-site component was less or equal to 0.10 log IU/mL.
The results are summarized in Table 6 and Table 7.
ot
-N

Table 6
Abbott RealTime HBV
Reproducibility in Plasma
y

0.5 mL Sample Preparation Protocol

nl

Within-Run Between-Run Between-Lot Between-Site

O

Mean Conc. Mean Conc. Component Component Component Component Total

Panel Genotype n (Log IU/mL) (IU/mL) SDc SDc SDc SDc SDc,d
1 A 120 8.93 872,502,276 0.07 0.00 0.03 0.08 0.11
n

2 A 119a 6.84 7,087,010 0.04 0.03 0.06 0.05 0.09

io

3 A 120 4.70 52,574 0.09 0.02 0.06 0.10 0.15

4 A 120 2.81 665 0.05 0.02 0.06 0.08 0.12
at

5 A 110b 0.90e 27e 0.31 0.07 0.26 0.10 0.42

6 C 119a 8.64 446,037,175 0.07 0.01 0.04 0.07 0.11
m

7 C 120 6.83 6,922,148 0.06 0.01 0.06 0.05 0.10

8 C 119a 4.84 72,954 0.08 0.00 0.08 0.09 0.15
or

9 C 120 2.84 722 0.06 0.02 0.09 0.08 0.13

10 C 119b 1.13 26 0.29 0.00 0.22 0.00 0.37
nf

a
Invalid replicate not included.
b
Target not detected not included.
rI

c
Standard deviations (SD) are in log IU/mL.
d
The total precision includes within-run, between-run, between-lot, and between-site components of precision.
e
Concentration is below the assay LoD.
Fo

8
 Table 7
Abbott RealTime HBV
Reproducibility in Plasma
0.2 mL Sample Preparation Protocol
Within-Run Between-Run Between-Lot Between-Site
Mean Conc. Mean Conc. Component Component Component Component Total
Panel Genotype n (Log IU/mL) (IU/mL) SDb SDb SDb SDb SDb,c
1 A 48 8.99 1,019,710,342 0.07 0.00 0.13 0.00 0.15
2 A 48 6.87 7,526,185 0.04 0.05 0.09 0.00 0.12
3 A 48 4.70 52,678 0.06 0.08 0.14 0.00 0.17
4 A 48 2.83 716 0.06 0.06 0.13 0.00 0.16
5 A 37a 1.03d 21 0.40 0.00 0.18 0.02 0.44
6 C 48 8.64 451,101,262 0.05 0.05 0.12 0.00 0.14
7 C 48 6.85 7,255,246 0.05 0.05 0.11 0.00 0.13
8 C 48 4.83 71,717 0.08 0.07 0.14 0.00 0.18
9 C 48 2.84 738 0.08 0.04 0.15 0.00 0.18
10 C 46a 1.24 26 0.30 0.00 0.27 0.00 0.41
a
Target not detected not included.
b
Standard deviations (SD) are in log IU/mL.
c
The total precision includes within-run, between-run, between-lot, and between-site components of precision.

y
d
Concentration is below the assay LoD.

op
REPRODUCIBILITY IN SERUM

C
The serum reproducibility panel tested at each site consisted of a 42-member panel (14 unique panel members) that included seven concentration levels of one prevalent HBV
genotype and seven concentration levels of a second prevalent HBV genotype, repeated three times within the panel. The concentration levels targeted for the reproducibility
panels spanned the linear quantitation range of the assay and also included some members below the lower limit of quantitation. The HBV genotypes selected for the serum

d
reproducibility panels were genotypes that were recognized as prevalent in the US population. Each seven-member panel was prepared from a high copy source sample. A total of

lle
three reagent lots were used. Each of the three clinical sites tested two of the three amplification reagent lots for five days each. Site 1 used lots A and B, Site 2 used lots B and
C, and Site 3 used lots A and C. Each site conducted the five day reproducibility at both the 0.2 mL volume and 0.5 mL volume for two lots of amplification reagents. The results
are summarized in Table 8 and Table 9.

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Table 8
Abbott RealTime HBV

on
Reproducibility in Serum
0.5 mL Sample Preparation Protocol

C
Within-Run Between-Run Between-Lot Between-Site
Mean Conc. Mean Conc. Component Component Component Component Total
a
Panel Genotype n (Log IU/mL) (IU/mL) SDc SDc SDc SDc SDc,d
1 A 89a 8.24 184,508,108 0.13 0.07 0.07 0.06 0.17
ot
2 A 90 6.19 1,613,319 0.04 0.07 0.07 0.03 0.11
3 A 90 3.94 9,725 0.07 0.08 0.11 0.19 0.24
-N

4 A 89a 1.96 105 0.12 0.07 0.07 0.20 0.25

5 A 90 1.25 22 0.20 0.10 0.12 0.20 0.32
6 A 88b 1.04 13 0.17 0.10 0.08 0.18 0.28
7 A 84b 0.74e 7e 0.22 0.12 0.21 0.12 0.35
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8 C 90 7.22 17,211,265 0.05 0.05 0.08 0.05 0.12

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9 C 90 6.23 1,741,264 0.05 0.06 0.08 0.02 0.12

10 C 89a 3.89 9,068 0.11 0.07 0.10 0.21 0.27
O

11 C 89a 1.64 55 0.18 0.12 0.06 0.28 0.36

12 C 90 1.29 23 0.18 0.09 0.12 0.15 0.27
13 C 88b 0.95e 12e 0.24 0.08 0.15 0.24 0.38
n

14 C 87b 0.88e 9e 0.23 0.06 0.10 0.10 0.28

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a
Invalid replicate not included.
b
Target not detected not included.
at

c
Standard deviations (SD) are in log IU/mL.
d
The total precision includes within-run, between-run, between-lot, and between-site components of precision.
m

e
Concentration is below the assay LoD.
or

Table 9
Abbott RealTime HBV
nf

Reproducibility in Serum
0.2 mL Sample Preparation Protocol
rI

Within-Run Between-Run Between-Lot Between-Site

Mean Conc. Mean Conc. Component Component Component Component Total
Fo

Panel Genotype n (Log IU/mL) (IU/mL) SDc SDc SDc SDc SDc,d
1 A 90 8.28 205,545,691 0.16 0.04 0.03 0.00 0.17
2 A 88a 6.21 1,638,140 0.04 0.04 0.03 0.00 0.06
3 A 89a 3.89 8,306 0.10 0.07 0.07 0.13 0.19
4 A 89a 1.90 95 0.22 0.04 0.16 0.16 0.32
5 A 88b 1.10e 16e 0.21 0.12 0.07 0.18 0.31
6 A 86a,b 0.89e 10e 0.27 0.03 0.10 0.10 0.31
7 A 82b 0.62e 7e 0.33 0.00 0.06 0.16 0.37
8 C 90 7.22 16,648,045 0.05 0.02 0.04 0.00 0.07
9 C 90 6.25 1,776,403 0.04 0.04 0.04 0.00 0.07
10 C 90 3.84 7,550 0.12 0.07 0.07 0.13 0.20
11 C 90 1.57 48 0.18 0.10 0.02 0.31 0.37
12 C 89b 1.14e 17e 0.22 0.10 0.11 0.12 0.29
13 C 87a,b 0.80 e
8e
0.26 0.13 0.15 0.08 0.34
14 C 82b 0.73e 9e 0.35 0.06 0.17 0.00 0.39
a
Invalid replicate not included.
b
Target not detected not included.
c
Standard deviations (SD) are in log IU/mL.
d
The total precision includes within-run, between-run, between-lot, and between-site components of precision.
e
Concentration is below the assay LoD.
9
LIMIT OF DETECTION (LoD) Using the WHO International Standard Table 13
The LoD of the Abbott RealTime HBV assay is 10 IU/mL for the 0.5 mL sample Abbott RealTime HBV
preparation protocol and 15 IU/mL for the 0.2 mL sample preparation protocol. The LoD Limit of Detection (LoD) in Serum Using the WHO International Standard
is defined as the HBV DNA concentration detected with a probability of 95%. The LoD 0.2 mL Sample Preparation Protocol
was determined by testing dilutions of the WHO International Standard for Hepatitis
B Virus DNA (NIBSC 97/746), which were prepared in HBV negative human plasma Number
and serum. Probit analysis of the data was used to determine the concentration of the IU/mL Tested Number Detected Percent Detected
WHO Standard detected with 95% probability. The results of the LoD study in plasma
and serum at both sample volumes are summarized in Tables 10 through 13. 40.00 30 30 100
20.00 30 30 100
10.00 30 30 100
Table 10 5.00 29a 25 86
Abbott RealTime HBV 2.50 30 27 90
Limit of Detection (LoD) in Plasma Using the WHO International Standard 1.00 30 17 57
0.5 mL Sample Preparation Protocol 0.50 30 17 57
0.20 30 4 13
Number
IU/mL Tested Number Detected Percent Detected
a
One replicate was excluded due to instrument error.
20.00 26 26 100
Probit analysis23 of the data determined that the concentration of HBV DNA detected
10.00 26 25 96 with 95% probability using the WHO International Standard was 5.61 IU/mL (95% CI

y
5.00 26 26 100 3.62-10.94 IU/mL).

op
2.50 26 23 88
LIMIT OF DETECTION (LoD) BY GENOTYPE USING CLINICAL SPECIMENS
1.00 26 12 46
The LoD for the assay to detect HBV in clinical specimens using the 0.5 mL sample
0.50 26 7 27

C
preparation protocol volume, detecting any of the eight genotypes tested, considering
0.25 26 7 27 that assay does not differentiate between HBV genotypes, was determined to be
0.10 26 4 15 10 IU/mL. The LoD for the assay to detect HBV in clinical specimens using the 0.2 mL

d
sample preparation protocol volume was determined to be 15 IU/mL.
Probit analysis23 of the data determined that the concentration of HBV DNA detected

lle
with 95% probability using the WHO International Standard was 6.40 IU/mL (95% CI The LoD was determined by analysis of a dilution series of patient samples
3.97-13.03 IU/mL). representing HBV Genotypes A, B, C, D, E, F, G, and H and of the WHO International
Standard. One patient sample for each HBV genotype was tested. Serial dilutions

tro
Table 11 were made in HBV serologically negative human plasma and serum to create an eight-
Abbott RealTime HBV member panel with the target concentrations 0.10 IU/mL, 0.25 IU/mL, 0.50 IU/mL,

 
1.00 IU/mL, 2.50 IU/mL, 5.00 IU/mL, 10.0 IU/mL, and 20.0 IU/mL for the 0.5 mL sample

on
Limit of Detection (LoD) in Serum Using the WHO International Standard

 
0.5 mL Sample Preparation Protocol volume and 0.20 IU/mL, 0.50 IU/mL, 1.00 IU/mL, 2.50 IU/mL, 5.00 IU/mL, 10.0 IU/mL,
20.0 IU/mL and 40.0 IU/mL for the 0.2 mL sample volume. Probit analysis of the data

C
Number was used to determine the concentration of each HBV genotype detected with 95%
IU/mL Tested Number Detected Percent Detected probability. The results are summarized in Table 14 and Table 15.
20.00 30 30 100
a Table 14
10.00 30 30 100
ot
5.00 30 30 100 Abbott RealTime HBV
2.50 30 29 97 LoD by Genotype Using Clinical Specimens
-N

1.00 30 17 57 0.5 mL Sample Preparation Protocol (IU/mL)

0.50 30 16 53 Genotype Concentration Detected (95% Confidence Interval)
0.25 30 1 3 Tested Plasma Serum
0.10 30 8 27
y

WHO 3.69 (2.59, 6.19) *

A 2.31 (1.59, 4.08) 5.49 (2.86, 19.05)
nl

Probit analysis23 of the data determined that the concentration of HBV DNA detected
with 95% probability using the WHO International Standard was 3.82 IU/mL (95% CI B 2.96 (2.12, 4.90) **
O

1.55-69.76 IU/mL). C 4.53 (3.18, 7.58) 3.92 (2.09, 14.50)

D 3.23 (2.23, 5.62) **
Table 12 E 4.73 (2.11, 39.36) 3.72 (2.55, 6.47)
n

Abbott RealTime HBV F 4.22 (2.80, 7.73) **

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Limit of Detection (LoD) in Plasma Using the WHO International Standard G 2.51 (1.80, 4.21) 1.94 (1.43, 3.14)
0.2 mL Sample Preparation Protocol H 8.11 (4.18, 27.97) **
at

* WHO standard was not tested in serum in this study.

Number ** Genotypes B, D, F, and H were tested in serum with 0.2 mL volume only. See Table 15.
m

IU/mL Tested Number Detected Percent Detected

40.00 27 27 100 The LoD for the assay to detect HBV in clinical specimens using the 0.5 mL sample
or

20.00 27 27 100 preparation protocol volume, detecting any of the eight genotypes tested, considering
10.00 27 26 96 that assay does not differentiate between HBV genotypes, was determined to be
nf

5.00 27 23 85 10 IU/mL.
2.50 27 12 44 Table 15
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1.00 27 11 41 Abbott RealTime HBV

0.50 27 6 22 LoD by Genotype Using Clinical Specimens
Fo

0.20 27 0 0 0.2 mL Sample Preparation Protocol (IU/mL)

Probit analysis23 of the data determined that the concentration of HBV DNA detected Genotype Concentration Detected (95% Confidence Interval)
with 95% probability using the WHO International Standard was 10.66 IU/mL (95% CI Tested Plasma Serum
7.11-19.38 IU/mL). WHO 8.16 (5.63, 13.93) *
A 5.86 (4.00, 10.22) **
B 5.37 (3.72, 9.23) 2.40 (1.61, 4.65)
C 8.61 (5.95, 14.68) **
D 5.34 (3.54, 9.93) 2.26 (1.55, 4.21)
E 14.57 (9.63, 26.28) **
F 6.60 (4.41, 11.98) 7.18 (4.75, 13.20)
G 3.84 (2.61, 6.94) **
H 10.86 (7.34, 19.10) 7.65 (5.01, 14.26)
* WHO standard was not tested in serum in this study.
** Genotypes A, C, E, and G were tested in serum with 0.5 mL volume only. See Table 14.

10
The LoD for the assay to detect HBV in clinical specimens using the 0.2 mL sample Microorganism/Virus Source
preparation protocol volume, detecting any of the eight genotypes tested, considering Human immunodeficiency virus 1 (HIV-1) Viral lysate, cell culture
that assay does not differentiate between HBV genotypes, was determined to be
Human immunodeficiency virus 2 (HIV-2) Viral lysate, cell culture
15 IU/mL.
Human T-lymphotropic virus I (HTLV-I) Viral lysate, cell culture
ANALYTICAL SPECIFICITY Hepatitis C virus (HCV) Viral lysate, human specimen
Potentially Interfering Substances Hepatitis A virus (HAV) Purified nucleic acid
The susceptibility of the Abbott RealTime HBV assay to interference by elevated Epstein-Barr virus (EBV) Purified nucleic acid
levels of potentially interfering substances was evaluated. HBV-negative samples and Herpes simplex virus 1 (HSV-1) Purified nucleic acid
HBV positive samples containing 2,933 IU/mL (3.47 log IU/mL) of HBV DNA were Herpes simplex virus 2 (HSV-2) Purified nucleic acid
‑
tested. Potential interference at HBV DNA concentrations close to the assay LLQ Cytomegalovirus (CMV) Purified nucleic acid
(LLoQ) was not assessed. HBV-negative and positive samples were tested in a plasma Human herpesvirus 6B (HHV-6B) Purified nucleic acid
matrix and were not tested in serum.
Human herpesvirus 8 (HHV-8) Purified nucleic acid
No interference in the performance of the Abbott RealTime HBV assay was observed in
Varicella-zoster virus (VZV) Purified nucleic acid
the presence of the following endogenous substances for all HBV-negative and positive
samples tested: Vaccinia virus (VACV) Purified nucleic acid
BK human polyomavirus Purified nucleic acid
• Hemoglobin 500 mg/dL Human papilloma virus 16 (HPV-16) Purified nucleic acid
• Triglycerides 3,000 mg/dL Human papilloma virus 18 (HPV-18) Purified nucleic acid
• Bilirubin 20 mg/dL Neisseria gonorrhoeae Purified nucleic acid
• Protein 9 g/dL Chlamydia trachomatis Purified nucleic acid

y
Note: For hemoglobin and protein, there was a slight trend toward lowering of the Candida albicans Purified nucleic acid

op
values of the high level HBV specimens in the presence of interfering substances. The Staphylococcus aureus Purified nucleic acid
mean differences of the test and control conditions for hemoglobin and protein are Staphylococcus epidermidis Purified nucleic acid
small (-0.058 and -0.112 log IU/mL, respectively) compared to the clinically significant Mycobacterium gordonae Purified nucleic acid

C
difference between two samples (1 log); as such, these differences are not expected to
Mycobacterium smegmatis Purified nucleic acid
be clinically significant.

d
Antivirals and antibiotics at concentrations equal to or in excess of peak plasma or
serum levels were tested in five pools. No interference in the performance of the Abbott Performance of the Assay with HBV-Negative Specimens

lle
RealTime HBV assay was observed in the presence of the following drug pools for all Performance of the Abbott RealTime HBV assay was evaluated by testing 124 HBV
HBV-negative and positive samples tested: serologically-negative serum and 125 HBV serologically-negative plasma specimens

tro
from blood donors. The specimens were tested on one m2000 instrument system with
Drug Pool Drugs Tested
one lot of amplification reagents. HBV DNA was not detected for all 249 specimens,
1 Zidovudine, Saquinavir, Ritonavir, Clarithromycin, Interferon 2a, resulting in 100% correct results: 100% (124/124) with 95% CI: 97.0% to 100% for serum

on
Interferon 2b, Didanosine samples and 100% (125/125) with 95% CI: 97.0% to 100% for plasma samples.
2 Abacavir sulfate, Amprenavir, Peginterferon 2a, Peginterferon 2b, ANALYTICAL CARRYOVER
Ribavirin, Entecavir, Adefovir Potential carryover was determined by performing three studies in which high copy

3 Tenofovir, Lamivudine, Indinavir, Ganciclovir, Valganciclovir, Acyclovir,
Paroxetine
4 Stavudine, Efavirenz, Lopinavir, Enfuvirtide, Ciprofloxacin, Fluoxetine
ot
5 Zalcitabine, Nevirapine, Nelfinavir, Azithromycin, Valacyclovir, Sertraline
Note: A consideration was made to avoid combining specific drugs within a pool that
-N
C
HBV positive samples were interspersed with negative samples in a checkerboard
‑
pattern. For these studies, the targeted level for the high copy HBV-positive samples
a
was greater than 8 log IU/mL. The carryover rate in these representative studies ranged
from 0% to 2%. For results greater or equal to LoD, there was an overall carryover rate
of 0.63% (95% CI 0.08%-2.24%). Results from the three studies are summarized in
Table 16.

would not be used together in a clinical setting. Because the listed drugs were tested in Table 16
pools, individual drug effects were not assessed.
Abbott RealTime HBV
Analytical Carryover
Cross-Reactivity Studies with Clinical Specimens
y

The specificity of the assay was evaluated by testing 60 patient specimens that were Number of Number Percent 95% CI of
nl

Number of Negatives Number Percent Detected Detected Percent

positive for at least one of the following DNA virus markers, RNA viruses, non-viral Study Runs Tested Detected Detected ( > LoD) ( > LoD) Detected
O

hepatitis, or autoimmune disease states. Specimens that were tested for DNA virus
markers were in serum. Specimens that were tested for RNA virus markers were in 1 5 100 1 1.00 0 0.00 (0.00, 3.62)
plasma or serum. HBV DNA was not detected in any of the 60 specimens tested. 2 5 100 2 2.00 2 2.00 (0.24, 7.04)
n

DNA and RNA Viruses Non-viral Hepatitis and Autoimmune States 3 6 120 2 1.67 0 0.00 (0.00, 3.03)
io

Epstein Barr Virus (EBV) Anti-nuclear Antibody (ANA)

Overall 16 320 5 1.56 2 0.63 (0.08, 2.24)
Herpes Simplex Virus 1 (HSV-1) Rheumatoid Factor (RF)
at

Herpes Simplex Virus 2 (HSV-2) Cirrhosis

Cytomegalovirus (CMV) Alcoholic Hepatitis COMPARISON OF 0.2 mL VS. 0.5 mL SAMPLE PREPARATION PROTOCOLS
m

Human Immunodeficiency Virus (HIV-1) Non-alcoholic Steatohepatitis (NASH) This study used the Abbott RealTime HBV assay to quantitate HBV-positive patient
or

Hepatitis C Virus (HCV) Autoimmune Hepatitis (AUH) specimens. Sixty HBV-positive EDTA plasma specimens were tested in duplicate with
Hepatitis A Virus (HAV) Hepatocellular Carcinoma both the 0.2 mL and 0.5 mL sample preparation protocols. Each duplicate pair was
 
tested in the same run. The data showed a slope of 0.99 and an intercept of 0.09. The
nf

Cross-Reactivity Studies Using Nucleic Acid or Viral Lysate study was designed to cover the dynamic range of the Abbott RealTime HBV assay with
The following viruses and microorganisms were evaluated for potential cross-reactivity actual patient samples representing genotypes (A, B, C, and D) commonly encountered
rI

in the Abbott RealTime HBV assay. Purified nucleic acid or viral lysate from each within the US.
microorganism or virus was added at a concentration of 100,000 copies/mL to The observed lowest value in the specimen population for the 0.2 mL sample volume
Fo

HBV DNA negative samples and HBV DNA positive samples that contained was 1.33 log IU/mL (mean value of the duplicate pair was 1.51 log IU/mL). For the
 
2,933 IU/mL (3.47 log IU/mL) HBV DNA. No interference in the performance of the 0.5 mL sample volume, the same specimen had an observed lowest value of
Abbott RealTime HBV assay was observed in the presence of the potential cross- 1.40 log IU/mL (mean value of the duplicate pair was 1.46 log IU/mL). The results are
reactant microorganisms or viruses for all the positive and negative samples tested. summarized in Figure 6.

11
 Table 17
Figure 6
Abbott RealTime HBV
Abbott RealTime HBV Specimen Stability (Log IU/mL)
Comparison of 0.2 mL vs. 0.5 mL Sample Preparation Protocols
Baseline
y = 0.99x + 0.09 Test Condition Condition Mean
r = 0.999 Sample Type Test Condition Mean Mean Difference
n = 60
24-26 hours
3.781 3.722 0.059
at 28-32°C
Plasma
0.2 mL Mean Concentration (Log IU/mL)

72-74 hours
3.777 3.722 0.055
at 2-8°C
24-26 hours
3.871 3.844 0.027
at 28-32°C
Serum
72-74 hours
3.870 3.844 0.026
at 2-8°C
6-8 hours
3.863 3.866 -0.003
Plasma at 28-32°C
(Whole Blood) 6-8 hours

y
3.862 3.866 -0.004
at 2-8°C

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6-8 hours
3.823 3.628 0.195
Serum at 28-32°C

C
(Whole Blood) 6-8 hours
3.730 3.628 0.102
at 2-8°C
0.5 mL Mean Concentration (Log IU/mL)

d
Plasma 8 freeze/thaw
2.704 2.693 0.011

lle
Freeze/Thaw cycles (frozen
SERUM VS. PLASMA ACROSS THE LINEAR RANGE at -20°C or
This study was conducted with specimens from 30 individual HBV serologically- colder for a

tro
negative donors. The specimens from each donor were collected as matched sets in minimum of 8
serum and in EDTA-plasma tubes. Each pair of serum and plasma specimens was Serum hours;
spiked with HBV-positive material at two targeted concentration levels throughout the thawed at 2.722 2.714 0.007

on
Freeze/Thaw
dynamic range of the Abbott RealTime HBV Assay. Specimen types at each targeted 15°C to 30°C
concentration were tested once using the 0.5 mL sample preparation protocol. Two for a maximum

C
plasma-serum pairs had quantitation values below the assay dynamic range and were of 24 hours)
therefore excluded from the analysis.
Using a sample size of 58, linear regression analysis demonstrated a slope of 1.00 a
(95% CI 0.98 to 1.01) and an intercept of 0.03 (95% CI -0.04 to 0.10). The mean CLINICAL STUDIES
difference between serum and plasma specimens was -0.02 log IU/mL (95% CI -0.05 to Study Population
ot
0.01). The linear regression is shown in Figure 7. The clinical performance of the Abbott RealTime HBV Assay for use with the
Figure 7 m2000 System was evaluated by assessing the antiviral therapy response in chronic
-N

Abbott RealTime HBV HBV infected subjects undergoing treatment with adefovir dipivoxil. The HBV DNA
‑
Serum vs. Plasma Across the Linear Range data were obtained from testing patient samples previously collected under two study
protocols, one of which evaluated patients with chronic HBeAg-positive HBV infection
and compensated liver function24 and one that evaluated patients with HBeAg-negative
y

HBV infection with compensated liver function.25 The relationship between HBV DNA
nl

viral levels at various time points to histologic, biochemical, and serological responses
to treatment was determined in this study.
O

The study population consisted of chronic HBV-infected patients enrolled in

double blind, randomized, placebo-controlled studies of adefovir dipivoxil that spanned
n

‑
240 weeks. In the HBeAg-positive protocol, patients were randomized to 10 mg
adefovir dipivoxil, 30 mg adefovir dipivoxil, or placebo for the first 48 weeks. Only the
io

10 mg adefovir dipivoxil treated patients (169 out of 171 total) and placebo patients
(60 randomly selected from 167 total) were included in this study. Viral load testing
at

was performed at baseline and at Weeks 12, 24, and 48. The viral load results were
evaluated against histologic, biochemical, and serological response at 48 weeks. In
m

addition, patients that remained on the 10 mg adefovir dipivoxil treatment were also
tested at Weeks 144, 192, 240, as available.
or

In the HBeAg-negative protocol, patients were randomized to either 10 mg adefovir

dipivoxil or placebo for the first 48 weeks. The adefovir dipivoxil treated patients (123
nf

out of 123 total) and placebo patients (61 out of 61 total) were tested at baseline
and at Weeks 12, 24, and 48. The viral load results were evaluated against histologic
rI

and biochemical response at 48 weeks. In addition, patients that remained on the 10

mg adefovir dipivoxil treatment were also tested at Weeks 96, 144, 192, and 240, as
Fo

available.
Demographic data, HBV genotype, HBeAg, anti-HBe, and HBsAg seroconversion results,
STABILITY and baseline (pretreatment) and post-treatment liver biopsy results were available. Table
18 summarizes the subject demographics.
Specimen Stability
Human serum or plasma specimens may be stored at 15 to 30°C for up to 24 hours
or at 2 to 8°C for up to three days. Freshly drawn whole blood (plasma or serum)
specimens may be held for up to 6 hours at 2 to 30 °C prior to centrifugation.
Freeze/thaw effect was tested in both serum and plasma for up to eight cycles. Frozen
specimens may be thawed at 15 to 30°C or 2 to 8°C. Thawed specimens may be
stored at 2 to 8°C for up to 6 hours, if not processed immediately. Serum and plasma
specimens may be stored at -20°C or colder for longer term storage. Stability testing
results are summarized in Table 17.

12
 Table 18 HBeAg-Positive Patients
Subject Demographics Characterization of Viral Load
Summary Table 20 and Figure 8 illustrate the efficacy, based on HBV viral load testing, of treating
Characteristic Category HBeAg+ HBeAg- Total
Statistics HBeAg-positive patients with 10 mg adefovir dipivoxil compared to placebo based on
Total Number of HBV viral load testing results using Abbott RealTime HBV assay for use with the m2000
- N 229 184 413
Subjects System. At Week 48, 22.92% (33/144) of HBeAg-positive patients on treatment versus
• Placebo - n (%) 60a (26.20) 61 (33.15) 121 0% (0/55) on placebo had achieved very low viral loads below 100 IU/mL. In addition,
• 10 mg adefovir only 23.61% (34/144) of patients on treatment versus 81.82% (45/55) on placebo had
- n (%) 169a (73.80) 123 (66.85) 292 viral loads greater than or equal to 106 IU/mL.
dipivoxil
Total Number
of Subjects with Table 20
- n 220 184 404
Demographic
Distribution of HBV Viral Load at Week 48 for HBeAg-Positive Patients
Information
Median 34 46 40 Adefovir Dipivoxil Placebo
Age (yr) - Viral Load Cumulative Cumulative
(Min, Max) (16, 65) (18, 65) (16, 65) n % n %
Median 71 74.55 72.5 (IU/mL) % %
Weight (kg) - TNDa 4 2.78 2.78 0 0.0 0.0
(Min, Max) (43, 117.73) (46, 135) (43, 135)
Male n (%) 164 (74.55) 152 (82.61) 316 (78.22) < 15 13 9.03 11.81 0 0.0 0.0
Sex 15 - <100 16 11.11 22.92 0 0.0 0.0
Female n (%) 56 (25.45) 32 (17.39) 88 (21.78)
100 - < 103 21 14.58 37.50 2 3.64 3.64

y
White n (%) 80 (36.36) 122 (66.30) 202 (50.00)
Race 103 - < 104 18 12.50 50.00 1 1.82 5.46

op
Asian n (%) 129 (58.64) 56 (30.43) 185 (45.79)
Other n (%) 11 (5.00) 6 (3.26) 17 (4.21) 104 - < 105 14 9.72 59.72 4 7.27 12.73
A n (%) 64 (29.09) 11 (5.98) 75 (18.56) 105 - < 106 24 16.67 76.39 3 5.45 18.18

C
B n (%) 41 (18.64) 31 (16.85) 72 (17.82) 106 - < 109 32 22.22 98.61 43 78.18 96.36
Genotype C n (%) 82 (37.27) 24 (13.04) 106 (26.24) ≥ 109 2 1.39 100.00 2 3.64 100.00

d
D n (%) 27 (12.27) 114 (61.96) 141 (34.90) Total 144 100.00 55 100.00
a
TND = Target Not Detected

lle
Other n (%) 6 (2.73) 4 (2.17) 10 (2.48)
Total Number Figure 8 demonstrates the median viral load change and inter-quartile range of change
of Subjects with - n 210 175 385 from baseline for HBeAg-positve subjects on treatment compared to placebo. This

tro
Knodell Score shows the impact of adefovir dipivoxil treatment on the viral load of HBeAg-positive
Total - Mean (SD) 9.38 (3.29) 9.35 (3.34) 9.37 (3.31) patients with chronic hepatitis B.
Necroinflammatory - Mean (SD) 7.70 (2.71) 7.50 (2.75) 7.61 (2.73)

on
Fibrosis - Mean (SD) 1.67 (1.09) 1.86 (1.15) 1.76 (1.12) Figure 8
a
Demographic data were not provided for three placebo and six treatment subjects. Median and Inter-Quartile Range of Change in

C
HBV DNA from Baseline: HBeAg-Positive Subjects
The HBeAg-positive subjects were primarily Asian and HBV Genotypes A and C, while a
the HBeAg-negative subjects were primarily White and HBV Genotype D. Patients
included in the clinical performance analysis received either the standard 10 mg
ot
adefovir dipivoxil dosing or placebo. Table 19 summarizes subjects by treatment arm
and available specimens.
-N

Table 19
Summary of Subjects by Treatment Arm
No. of No. of No. of Total No. of
Total No. Subjects - Subjects - Specimens per Specimens
y

Population of Subjects Placebo 10 mg Adefovir Subjecta Tested

nl

Chronic HBeAg+ 229 60 169 2 to 7 1,036

Chronic HBeAg- 184 61 123 2 to 8 939
O

Total Number of
413 121 292 2 to 8 1,975
Subjects Tested
a
This number is reported as a range because the number of specimens varied for each subject.
n
io

CLINICAL STUDY RESULTS AND STATISTICAL ANALYSES

Statistical analysis of clinical data was used to assess whether viral response to
at

treatment measured with Abbott RealTime HBV Assay for use with the m2000 System
is informative for determining the response to treatment in HBeAg-positive and
m

HBeAg negative patients with chronic hepatitis B. Observing changes in viral load in The effect of therapy for patients with chronic HBV infection can be assessed by
‑
individual patients over time may help the clinician in the assessment of a patient’s measuring the HBV DNA (expected reduction to low or undetectable levels), and
or

response to therapy. monitoring for viral rebound that could be associated with resistance. Results in Table
21 show that 70.41% (119/169) of the treated subjects achieved a nadir, or lowest
nf

Within-Subject Variability in Absence of Treatment concentration, viral load level by Week 48. Of the 49 subjects that achieved a nadir by
The objective of this analysis was to assess the change in viral load (in log IU/mL units) Week 24, 13.79% (4/29) of the subjects had a greater than or equal to one log IU/mL
rI

between two successive measurements of placebo patients. There were 55 patients in increase by Week 48 (20 of these subjects did not have a Week 48 result).
the placebo arm of the HBeAg-positive group and 57 patients in the HBeAg-negative
Fo

group that had available results for both Weeks 0 and 12. These results were used to
estimate within-subject variability, which includes biological variability as well as total
assay variability.
The within-subject variability (SD) based on these results was estimated to be
0.79 log IU/mL for HBeAg-positive patients and 0.86 log IU/mL for HBeAg-negative
patients. Biological within-subject variability was similar to the estimated within-subject
variability since the assay analytical variability was negligible. The median change
(Week 12 – Week 0) of viral load within a subject was estimated to be
0.00 log IU/mL for HBeAg-positive patients and -0.28 log IU/mL for HBeAg-negative
 
 
 
patients. Approximately 89% of the HBeAg-positive patients’ and 81% of HBeAg-
negative patients’ change of viral load was less than 2.00 log IU/mL.
 
13
 Table 21
Distribution of HBeAg-Positive Subjects by Week on
Treatment and the Viral Load at Which the Nadir was Reached

Number (%) of Patients With the Nadir Viral Load Achieved by Week
Nadir Viral Load Total By Cumulative
(IU/mL) 12 24 48 144 192 240 Viral Load By Viral Load
TNDa 0 (0.00) 0 (0.00) 4 (2.37) 1 (0.59) 1 (0.59) 4 (2.37) 10 (5.92) 10 (5.92)
< 15 0 (0.00) 1 (0.59) 13 (7.69) 1 (0.59) 6 (3.55) 1 (0.59) 22 (13.02) 32 (18.94)
15 - < 100 0 (0.00) 0 (0.00) 12 (7.10) 3 (1.78) 4 (2.37) 2 (1.18) 21 (12.43) 53 (31.37)
100 - < 10 3
1 (0.59) 3 (1.78) 15 (8.88) 0 (0.00) 1 (0.59) 7 (4.14) 27 (15.98) 80 (47.35)
103 - < 104 3 (1.78) 8 (4.73) 5 (2.96) 1 (0.59) 1 (0.59) 2 (1.18) 20 (11.83) 100 (59.18)
104 - < 105 3 (1.78) 5 (2.96) 7 (4.14) 3 (1.78) 1 (0.59) 2 (1.18) 21 (12.43) 121 (71.61)
10 - < 10
5 6
3 (1.78) 5 (2.96) 8 (4.73) 1 (0.59) 3 (1.78) 2 (1.18) 22 (13.02) 143 (84.63)
106 - < 109 8 (4.73) 9 (5.33) 6 (3.55) 1 (0.59) 0 (0.00) 2 (1.18) 26 (15.38) 169 (100.00)
Total By Week 18 (10.65) 31 (18.34) 70 (41.42) 11 (6.51) 17 (10.06) 22 (13.02)

y
Cumulative By Week 18 (10.65) 49 (28.99) 119 (70.41) 130 (76.92) 147 (86.98) 169 (100.00)

op
a
TND = Target Not Detected

Two patients out of 169 achieved HBsAg seroconversion. One patient had results showing HBsAg serconversion at both Weeks 192 and 240. The other patient achieved

C
seroconversion at Week 240. These two patients were white males, HBV genotype A, and > 30 years of age. A summary of these results is provided in Table 22.

d
Table 22

lle
HBeAg-Positive Subjects with HBsAg Seroconversion

tro
Concentration (Log IU/mL)
Week 0 Week 12 Week 24 Week 48 Week 144 Week 192 Week 240

on
Subject 1 6.99 4.98 2.08 1.50 TNDa TNDa TNDa
Subject 2 8.59 6.57 6.85 6.72 5.68 1.45 *

C
a
TND = Target Not Detected
* The Abbott RealTime HBV result for the Week 240 time point was excluded due to technician error.
a
Summaries of the effect of baseline covariates for the HBeAg-positive population are provided in Table 23 through Table 25.
ot
Table 23
-N

Association Between Responses to Treatment at Week 48 and Baseline Covariates

for HBeAg-Positive Patients
No. of Proportion (%)
y

Response to Patients with of Patients with Unadjusted Odds

Treatment Covariate Category N Response Response Ratio (95% CI)
nl

Asian 75 47 62.67
Race 1.44 (0.66, 3.14)
Other 52 28 53.85
O

Male 97 58 59.79
Sex 1.14 (0.45, 2.81)
Female 30 17 56.67
Histological
n

≤ 30 52 34 65.38
Age 1.57 (0.71, 3.49)
> 30 75 41 54.67
io

B,C 72 47 65.28
Genotype 1.81 (0.83, 3.95)
Non-B,C 55 28 50.91
at

Asian 82 47 57.32
Race 1.77 (0.82, 3.82)
Other 51 22 43.14
m

Male 102 53 51.96

Sex 1.01 (0.42, 2.45)
Female 31 16 51.61
or

Biochemical
≤ 30 56 33 58.93
Age 1.63 (0.77, 3.48)
> 30 77 36 46.75
nf

B,C 79 45 56.96
Genotype 1.65 (0.78, 3.53)
Non-B,C 54 24 44.44
rI

Asian 84 21 25.00
Race 0.89 (0.38, 2.09)
Other 55 15 27.27
Male 105 26 24.76
Fo

Sex 0.79 (0.31, 2.11)

Female 34 10 29.41
HBeAg Loss
≤ 30 59 13 22.03
Age 0.70 (0.29, 1.63)
> 30 80 23 28.75
B,C 80 19 23.75
Genotype 0.77 (0.34, 1.78)
Non-B,C 59 17 28.81
Asian 84 8 9.52
Race 0.47 (0.15, 1.45)
Other 55 10 18.18
Male 105 14 13.33
Sex 1.15 (0.33, 5.18)
HBeAg Sero- Female 34 4 11.76
conversion ≤ 30 59 7 11.86
Age 0.84 (0.26, 2.58)
> 30 80 11 13.75
B,C 80 6 7.50
Genotype 0.32 (0.09, 1.00)
Non-B,C 59 12 20.34

14
The statistical significance of the associations of the Race, Sex, Age and Genotype covariates with viral response was studied, and the results are summarized in Tables 24 and
Table 25. All lower limits of the 95% confidence intervals in Table 24 are smaller than 1, except for Race and Genotype at Weeks 12 and 24 (when viral response is defined as
< 2,000 IU/mL). When response is defined as < 2,000 IU/mL, logistic regression analyses resulted in no statistically significant associations between the four covariates and
viral load. All lower limits of the 95% confidence intervals in Table 25 are smaller than 1 (when viral response is defined as ≥ 2 log decrease). When response is defined as ≥ 2
log decrease, logistic regression analyses resulted in only gender at Week 12 showing a borderline statistically significant association (p = 0.043) with viral load. Generally, the
virological responses at Weeks 12, 24 and 48 do not appear to be correlated with Race, Sex, Age, and HBV Genotype.

Table 24
Odds Ratios for the Association Between Viral Response (< 2,000 IU/mL) and Covariates, by Week,
for an HBeAg-Positive Population
No. Below Proportion (%) Unadjusted Odds
Covariate Category Week N 2,000 IU/mL Below 2,000 IU/mL Ratio (95% CI)
Asian 87 21 24.14
12 2.92 (1.04, 9.41)
Other 61 6 9.84
Asian 81 30 37.04
Race 24 3.20 (1.30, 8.42)
Other 58 9 15.52
Asian 79 34 43.04
48 1.56 (0.71, 3.47)
Other 52 17 32.69
Male 112 20 17.86
12 0.90 (0.32, 2.78)
Female 36 7 19.44

y
Male 104 27 25.96
Sex 24 0.67 (0.28, 1.70)

op
Female 35 12 34.29
Male 100 37 37.00
48 0.71 (0.29, 1.76)
Female 31 14 45.16

C
≤ 30 63 10 15.87
12 0.75 (0.28, 1.92)
> 30 85 17 20.00
≤ 30 61 18 29.51

d
Age 24 1.14 (0.50, 2.55)
> 30 78 21 26.92

lle
≤ 30 55 21 38.18
48 0.95 (0.44, 2.05)
> 30 76 30 39.47
B,C 81 20 24.69

tro
12 2.81 (1.04, 8.41)
Non-B,C 67 7 10.45
B,C 76 29 38.16
Genotype 24 3.27 (1.36, 8.29)
Non-B,C 63 10 15.87

on
B,C 74 32 43.24
48 1.52 (0.70, 3.34)
Non-B,C 57 19 33.33

C
Table 25
a
Odds Ratios for the Association Between Viral Response ( ≥ 2 Log Decrease From Baseline Result) and Covariates, by Week,
for an HBeAg-Positive Population
ot
No. with Proportion (%)
≥ 2 Log with ≥ 2 Log Unadjusted Odds
-N

Covariate Category Week N Decrease Decrease Ratio (95% CI)

Asian 87 54 62.07
12 0.86 (0.41, 1.79)
Other 61 40 65.57
Asian 81 62 76.54
y

Race 24 1.24 (0.53, 2.88)

Other 58 42 72.41
nl

Asian 79 56 70.89
48 0.81 (0.33, 1.91)
Other 52 39 75.00
O

Male 112 66 58.93

12 0.41 (0.15, 1.03)
Female 36 28 77.78
Male 104 74 71.15
n

Sex 24 0.41 (0.11, 1.22)

Female 35 30 85.71
io

Male 100 71 71.00

48 0.71 (0.23, 1.96)
Female 31 24 77.42
at

≤ 30 63 41 65.08
12 1.13 (0.54, 2.36)
> 30 85 53 62.35
m

≤ 30 61 44 72.13
Age 24 0.78 (0.34, 1.81)
> 30 78 60 76.92
or

≤ 30 55 40 72.73
48 1.02 (0.44, 2.41)
> 30 76 55 72.37
B,C 81 51 62.96
nf

12 0.95 (0.46, 1.96)

Non-B,C 67 43 64.18
B,C 76 58 76.32
rI

Genotype 24 1.19 (0.51, 2.75)

Non-B,C 63 46 73.02
B,C 74 52 70.27
Fo

48 0.77 (0.32, 1.80)

Non-B,C 57 43 75.44

Positive Predictive Value (PPV), Negative Predictive Value (NPV), and Odds Ratio (OR) Analysis in a HBeAg-Positive Population
For each patient, the clinical responses: Histologic, Biochemical, HBeAg Loss, Anti-HBe Gain, and Seroconversion were measured at various times on treatment. These clinical
responses were defined as follows:
• Histologic response - improvement of histologic status by at least 2 units of the Knodell necro-inflammatory score without deterioration of the fibrosis score compared to the
histologic status at baseline
• Biochemical response - normalization of ALT test result compared to the biochemical status at baseline
• HBeAg Loss - HBeAg undetectable
• Anti-HBe Gain - antibody against HBeAg detected
• Seroconversion - HBeAg undetectable and antibody against HBeAg detected

15
Additionally, HBsAg seroconversion data were collected. Two patients out of 169 achieved HBsAg seroconversion. One patient had results showing HBsAg serconversion at both
Weeks 192 and 240. The other patient achieved seroconversion at Week 240. These two patients were white males, HBV genotype A, and > 30 years of age. A summary of these
results is provided in Table 22.
Viral load response was defined as either HBV DNA less than 2,000 IU/mL or greater than or equal to 2 log IU/mL decrease from baseline. Statistical analysis (PPV) was
performed to evaluate the association between the clinical responses at Weeks 48, 144, 192, or 240 and a viral load response at Weeks 12, 24, or 48 of treatment. Statistical
analysis (NPV) was performed to evaluate whether there is an association between the clinical non-responses at Weeks 48, 144, 192, or 240 and a viral load non-response at
Weeks 12, 24, or 48 of treatment.
Viral Response < 2,000 IU/mL
As shown in Table 26, early viral response (Weeks 12, 24, 48) is informative in predicting clinical responses at Week 48. The PPV is the highest for the association of viral
response and the histologic and biochemical responses; while NPV is the highest for the association of viral response and the serological responses (HBeAg loss, anti-HBe gain,
and seroconversion).
Viral response at Weeks 12, 24, and 48 is informative in predicting biochemical, HBeAg loss, anti-HBe gain, and seroconversion at Week 48 (i.e., the lower 95% CI limits for the
odds ratio exceed 1.0). Viral response at Week 24 is also informative in predicting histologic improvement at Week 48. Viral response at Week 24 is informative in predicting HBeAg
loss at Week 144 and viral response at Week 48 is also informative in predicting anti-HBe gain and seroconversion at Week 240 of treatment.

Table 26
PPV, NPV, and Odds Ratio for Individual Clinical Responses During Treatment
Predicted by Early Viral Response (< 2,000 IU/mL) in HBeAg-Positive Subjects
Week of Viral Week of Clinical PPV NPV Odds Ratio
Clinical Response PPV (%) (Proportion) NPV (%) (Proportion)

y
Response Response (95% CI) (95% CI) (95% CI)a

op
Histologic 79.2 (19/24) (57.3, 92.1) 43.3 (39/90) (33.1, 54.2) 2.91 (0.93, 10.76)
Biochemical 84.0 (21/25) (63.1, 94.7) 55.3 (52/94) (44.7, 65.5) 6.50 (1.95, 27.66)
12 48 HBeAg Loss 64.0 (16/25) (42.6, 81.3) 82.8 (82/99) (73.6, 89.4) 8.58 (2.94, 25.54)

C
Anti-HBe Gain 36.0 (9/25) (18.7, 57.4) 91.9 (91/99) (84.2, 96.2) 6.40 (1.85, 21.90)
Seroconversionb 36.0 (9/25) (18.7, 57.4) 91.9 (91/99) (84.2, 96.2) 6.40 (1.85, 21.90)

d
Histologic 80.0 (28/35) (62.5, 90.9) 49.3 (37/75) (37.7, 61.0) 3.89 (1.42, 11.76)

lle
Biochemical 83.8 (31/37) (67.3, 93.2) 59.7 (46/77) (47.9, 70.6) 7.67 (2.68, 24.74)
24 48 HBeAg Loss 62.2 (23/37) (44.8, 77.1) 90.2 (74/82) (81.2, 95.4) 15.20 (5.15, 46.52)
Anti-HBe Gain 24.3 (9/37) (12.4, 41.6) 92.7 (76/82) (84.2, 97.0) 4.07 (1.16, 15.06)

tro
Seroconversion 24.3 (9/37) (12.4, 41.6) 92.7 (76/82) (84.2, 97.0) 4.07 (1.16, 15.06)
Histologic 74.0 (37/50) (59.4, 84.9) 49.3 (35/71) (37.3, 61.3) 2.77 (1.19, 6.62)

on
Biochemical 78.0 (39/50) (63.7, 88.0) 63.2 (48/76) (51.3, 73.7) 6.08 (2.52, 15.15)
48c 48 HBeAg Loss 62.7 (32/51) (48.1, 75.5) 97.5 (78/80) (90.4, 99.6) 65.68 (14.10, 587.82)
Anti-HBe Gain 33.3 (17/51) (21.1, 48.0) 100.0 (80/80) (94.3, 100.0) >39.50d

C
Seroconversion 33.3 (17/51) (21.1, 48.0) 100.0 (80/80) (94.3, 100.0) >39.50d
Histologic a not available
Biochemical 66.7 (4/6) (24.1, 94.0) 55.6 (25/45) (40.1, 70.0) 2.50 (0.31, 29.77)
12 144 HBeAg Loss 50.0 (3/6) (13.9, 86.1) 84.4 (38/45) (69.9, 93.0) 5.43 (0.58, 47.41)
ot
Anti-HBe Gain 33.3 (2/6) (6.0, 75.9) 86.7 (39/45) (72.5, 94.5) 3.25 (0.24, 28.53)
Seroconversion 33.3 (2/6) (6.0, 75.9) 86.7 (39/45) (72.5, 94.5) 3.25 (0.24, 28.53)
-N

Histologic not available

Biochemical 70.0 (7/10) (35.4, 91.9) 57.5 (23/40) (41.0, 72.6) 3.16 (0.60, 21.20)
24 144 HBeAg Loss 50.0 (5/10) (20.1, 79.9) 87.5 (35/40) (72.4, 95.3) 7.00 (1.11, 42.84)
y

Anti-HBe Gain 30.0 (3/10) (8.1, 64.6) 87.5 (35/40) (72.4, 95.3) 3.00 (0.37, 19.68)
Seroconversion 30.0 (3/10) (8.1, 64.6) 87.5 (35/40) (72.4, 95.3) 3.00 (0.37, 19.68)
nl

Histologic not available

O

Biochemical 63.6 (7/11) (31.6, 87.6) 58.3 (21/36) (40.9, 74.0) 2.45 (0.50, 13.33)
48 144 HBeAg Loss 36.4 (4/11) (12.4, 68.4) 86.1 (31/36) (69.7, 94.8) 3.54 (0.54, 21.21)
Anti-HBe Gain 27.3 (3/11) (7.3, 60.7) 88.9 (32/36) (73.0, 96.4) 3.00 (0.36, 21.45)
n

Seroconversion 27.3 (3/11) (7.3, 60.7) 88.9 (32/36) (73.0, 96.4) 3.00 (0.36, 21.45)
io

Histologic not available

Biochemical 60.0 (3/5) (17.0, 92.7) 39.5 (15/38) (24.5, 56.6) 0.98 (0.10, 13.02)
at

12 192 HBeAg Loss 40.0 (2/5) (7.3, 83.0) 57.9 (22/38) (40.9, 73.3) 0.92 (0.07, 9.02)
Anti-HBe Gain 40.0 (2/5) (7.3, 83.0) 76.3 (29/38) (59.4, 88.0) 2.15 (0.15, 21.62)
m

Seroconversion 40.0 (2/5) (7.3, 83.0) 76.3 (29/38) (59.4, 88.0) 2.15 (0.15, 21.62)
or

Histologic not available

Biochemical 66.7 (6/9) (30.9, 91.0) 40.6 (13/32) (24.2, 59.2) 1.37 (0.24, 9.92)
nf

24 192 HBeAg Loss 44.4 (4/9) (15.3, 77.3) 62.5 (20/32) (43.7, 78.3) 1.33 (0.22, 7.58)
Anti-HBe Gain 33.3 (3/9) (9.0, 69.1) 78.1 (25/32) (59.6, 90.1) 1.79 (0.23, 11.22)
rI

Seroconversion 33.3 (3/9) (9.0, 69.1) 78.1 (25/32) (59.6, 90.1) 1.79 (0.23, 11.22)
Histologic not available
Fo

Biochemical 77.8 (7/9) (40.2, 96.1) 37.5 (12/32) (21.7, 56.3) 2.10 (0.32, 23.57)
48 192 HBeAg Loss 66.7 (6/9) (30.9, 91.0) 62.5 (20/32) (43.7, 78.3) 3.33 (0.56, 23.78)
Anti-HBe Gain 55.6 (5/9) (22.7, 84.7) 81.3 (26/32) (63.0, 92.1) 5.42 (0.83, 35.32)
Seroconversion 55.6 (5/9) (22.7, 84.7) 81.3 (26/32) (63.0, 92.1) 5.42 (0.83, 35.32)
Histologic * (0/0) * 33.3 (3/9) (9.0, 69.1) *
Biochemical 66.7 (2/3) (12.5, 98.2) 29.0 (9/31) (14.9, 48.2) 0.82 (0.04, 53.57)
12 240 HBeAg Loss 66.7 (2/3) (12.5, 98.2) 58.1 (18/31) (39.3, 74.9) 2.77 (0.13, 172.28)
Anti-HBe Gain 33.3 (1/3) (1.8, 87.5) 86.7 (26/30) (68.4, 95.6) 3.25 (0.04, 73.98)
Seroconversion 33.3 (1/3) (1.8, 87.5) 87.1 (27/31) (69.2, 95.8) 3.38 (0.05, 76.68)

16
 Histologic 0.0 (0/1) (0.0, 94.5) 37.5 (3/8) (10.2, 74.1) 0.00 (0.00, 15.20)
Biochemical 60.0 (3/5) (17.0, 92.7) 29.6 (8/27) (14.5, 50.3) 0.63 (0.06, 9.06)
24 240 HBeAg Loss 60.0 (3/5) (17.0, 92.7) 63.0 (17/27) (42.5, 79.9) 2.55(0.24, 34.44)
Anti-HBe Gain 20.0 (1/5) (1.1, 70.1) 84.6 (22/26) (64.3, 95.0) 1.38 (0.02, 20.07)
Seroconversion 20.0 (1/5) (1.1, 70.1) 85.2 (23/27) (65.4, 95.1) 1.44 (0.02, 20.91)
Histologic 100.0 (1/1) (5.5, 100.0) 57.1 (4/7) (20.2, 88.2) *
Biochemical 83.3 (5/6) (36.5, 99.1) 30.8 (8/26) (15.1, 51.9) 2.22 (0.19, 118.05)
48 240 HBeAg Loss 83.3 (5/6) (36.5, 99.1) 65.4 (17/26) (44.4, 82.1) 9.44 (0.81, 472.23)
Anti-HBe Gain 50.0 (3/6) (13.9, 86.1) 96.0 (24/25) (77.7, 99.8) 24.00 (1.21, 1,309.0)
Seroconversion 50.0 (3/6) (13.9, 86.1) 96.2 (25/26) (78.4, 99.8) 25.00 (1.26, 1,361.3)
* Undefined (division by zero)
a
Shading indicates statistical significance.
b
Seroconversion - HBeAg undetectable and antibody against HBeAg detected
c
An association with, rather than prediction of, clinical responses is demonstrated when measuring the viral response at Week 48.
d
The odds ratio calculations are undefined when NPV is 100% or PPV is 100% or missing. Where the denominators for both the NPV and PPV are greater than five, a “minimum” odds ratio was
determined by subtracting one specimen from the numerator of the 100% parameter estimate (NPV or PPV).

Tables 27 and 28 demonstrate that the NPV is very high (greater than 89% for Week 48 of clinical response) for the association of early viral response with the combination of
all three responses (histologic, biochemical, and serological - HBeAg loss or seroconversion). These data indicate that HBeAg-positive subjects without an early viral response
(defined as < 2,000 IU/mL decrease) are very unlikely to achieve all three clinical responses by Week 48, as a result of treatment. The number of subjects at Week 240 was small,

y
and therefore available data is inadequate to draw conclusions about the association of the early viral response with clinical responses at later weeks.

op
Table 27
PPV, NPV, and Odds Ratio (OR) for a Combination of Histologic, Biochemical, and HBeAg Loss Responses During Treatment Predicted by an Early Viral

C
Response (< 2,000 IU/mL) in HBeAg-Positive Subjects
Week of Viral Week of Clinical PPV (%) (Proportion) PPV NPV Odds Ratio

d
Response Response (95% CI) NPV (%) (Proportion) (95% CI) (95% CI)a

lle
12 48 41.7 (10/24) (22.8, 63.1) 89.9 (80/89) (81.2, 95.0) 6.35 (1.90, 20.95)
24 48 42.9 (15/35) (26.8, 60.5) 97.3 (72/74) (89.7, 99.5) 27.00 (5.38, 252.97)

tro
48b 48 36.7 (18/49) (23.8, 51.7) 98.6 (70/71) (91.3, 99.9) 40.65 (5.75, 1716.7)
12 240 * (0/0) * 55.6 (5/9) (22.7, 84.7) *
24 240 0.0 (0/1) (0.0, 94.5) 62.5 (5/8) (25.9, 89.8) 0.00 (0.00, 38.00)

on
48 240 100.0 (1/1) (5.5, 100.0) 71.4 (5/7) (30.3, 94.9) *
* Undefined (division by zero)
a
Shading indicates statistical significance.

C
b
An association with, rather than prediction of, clinical responses is demonstrated when measuring the viral response at Week 48.

Table 28
a
PPV, NPV, and Odds Ratio (OR) for a Combination of Histologic, Biochemical, and Seroconversion Responses During Treatment Predicted by an Early Viral
ot
Response (< 2,000 IU/mL) in HBeAg-Positive Subjects
-N

Week of Viral Week of Clinical PPV (%) (Proportion) PPV NPV (%) (Proportion) NPV Odds Ratio
Response Response (95% CI) (95% CI) (95% CI)a
12 48 25.0 (6/24) (10.6, 47.1) 97.8 (87/89) (91.4, 99.6) 14.50 (2.28, 152.63)
24 48 17.1 (6/35) (7.2, 34.3) 98.6 (73/74) (91.7, 99.9) 15.10 (1.68, 703.89)
y

48b 48 16.3 (8/49) (7.8, 30.2) 100.0 (71/71) (93.6, 100.0) >13.66c
12 240 * (0/0) * 77.8 (7/9) (40.2, 96.1) *
nl

24 240 0.0 (0/1) (0.0, 94.5) 75.0 (6/8) (35.6, 95.5) 0.00 (0.00, 66.50)
48 240 100.0 (1/1) (5.5, 100.0) 100.0 (7/7) (56.1, 100.0) *
O

* Undefined (division by zero)

a
Shading indicates statistical significance.
n

b
An association with, rather than prediction of, clinical responses is demonstrated when measuring the viral response at Week 48.
c
The odds ratio calculations are undefined when NPV is 100% or PPV is 100% or missing. Where the denominators for both the NPV and PPV are greater than five, a “minimum” odds ratio
io

was determined by subtracting one specimen from the numerator of the 100% parameter estimate (NPV or PPV).
at

Viral Response ≥ 2 Log IU/mL Decrease

As shown in Table 29, early viral response (Weeks 12, 24, 48) is informative in predicting clinical responses at Week 48. High NPV (>90.9%) is observed for the association of viral
m

response and the serological responses (HBeAg loss, anti-HBe Gain, and seroconversion). The significance of a viral response at Weeks 12, 24, and 48 in predicting histologic,
biochemical, HBeAg loss, anti-HBe gain, and seroconversion at Week 48 and later time points is assessed by the lower 95% CI limit for the odds ratio exceeding 1.0.
or

Table 29
nf

PPV, NPV, and Odds Ratio for Individual Clinical Responses During Treatment
Predicted by Early Viral Response ( ≥ 2 Log IU/mL Decrease) in HBeAg-Positive Subjects
rI

Week of
Week of Viral PPV NPV Odds Ratio
Clinical Clinical Response PPV (%) (Proportion) NPV (%) (Proportion)
Fo

Response (95% CI) (95% CI) (95% CI)a

Response
Histologic 67.5 (52/77) (55.8, 77.5) 51.4 (19/37) (34.7, 67.8) 2.20 (0.91, 5.28)
Biochemical 58.2 (46/79) (46.6, 69.1) 57.5 (23/40) (41.0, 72.6) 1.89 (0.82, 4.39)
12 48 HBeAg Loss 36.3 (29/80) (26.0, 47.8) 90.9 (40/44) (77.4, 97.0) 5.69 (1.76, 23.79)
Anti-HBe Gain 18.8 (15/80) (11.2, 29.4) 95.5 (42/44) (83.3, 99.2) 4.85 (1.03, 45.38)
Seroconversionb 18.8 (15/80) (11.2, 29.4) 95.5 (42/44) (83.3, 99.2) 4.85 (1.03, 45.38)
Histologic 62.7 (52/83) (51.3, 72.8) 48.1 (13/27) (29.2, 67.6) 1.56 (0.59, 4.09)
Biochemical 59.3 (51/86) (48.2, 69.6) 60.7 (17/28) (40.7, 77.9) 2.25 (0.87, 5.98)
24 48 HBeAg Loss 33.3 (30/90) (24.0, 44.1) 96.6 (28/29) (80.4, 99.8) 14.00 (2.07, 591.20)
Anti-HBe Gain 16.7 (15/90) (9.9, 26.3) 100.0 (29/29) (85.4, 100.0) >5.60d
Seroconversion 16.7 (15/90) (9.9, 26.3) 100.0 (29/29) (85.4, 100.0) >5.60d
Histologic 66.7 (60/90) (55.9, 76.0) 58.1 (18/31) (39.3, 74.9) 2.77 (1.11, 7.00)
Biochemical 63.4 (59/93) (52.8, 73.0) 75.8 (25/33) (57.4, 88.3) 5.42 (2.06, 15.32)
48c 48 HBeAg Loss 35.8 (34/95) (26.4, 46.3) 100.0 (36/36) (88.0,100.0) >19.51d
Anti-HBe Gain 17.9 (17/95) (11.1, 27.4) 100.0 (36/36) (88.0,100.0) >7.63d
Seroconversion 17.9 (17/95) (11.1, 27.4) 100.0 (36/36) (88.0,100.0) >7.63d

17
 Histologic not available
Biochemical 43.3 (13/30) (26.0, 62.3) 47.6 (10/21) (26.4, 69.7) 0.70 (0.20, 2.46)
12 144 HBeAg Loss 23.3 (7/30) (10.6, 42.7) 85.7 (18/21) (62.6, 96.2) 1.83 (0.35, 12.35)
Anti-HBe Gain 16.7 (5/30) (6.3, 35.5) 85.7 (18/21) (62.6, 96.2) 1.20 (0.20, 8.70)
Seroconversion 16.7 (5/30) (6.3, 35.5) 85.7 (18/21) (62.6, 96.2) 1.20 (0.20, 8.70)
Histologic not available
Biochemical 44.7 (17/38) (29.0, 61.5) 41.7 (5/12) (16.5, 71.4) 0.58 (0.12, 2.59)
24 144 HBeAg Loss 26.3 (10/38) (14.0, 43.4) 100.0 (12/12) (69.9, 100.0) >3.93d
Anti-HBe Gain 21.1 (8/38) (10.1, 37.8) 100.0 (12/12) (69.9, 100.0) >2.93d
Seroconversion 21.1 (8/38) (10.1, 37.8) 100.0 (12/12) (69.9, 100.0) >2.93d
Histologic not available
Biochemical 48.3 (14/29) (29.9, 67.1) 55.6 (10/18) (31.3, 77.6) 1.17 (0.31, 4.49)
48 144 HBeAg Loss 20.7 (6/29) (8.7, 40.3) 83.3 (15/18) (57.7, 95.6) 1.30 (0.23, 9.25)
Anti-HBe Gain 17.2 (5/29) (6.5, 36.5) 88.9 (16/18) (63.9, 98.1) 1.67 (0.23, 19.34)
Seroconversion 17.2 (5/29) (6.5, 36.5) 88.9 (16/18) (63.9, 98.1) 1.67 (0.23, 19.34)
Histologic not available
Biochemical 60.0 (15/25) (38.9, 78.2) 38.9 (7/18) (18.3, 63.9) 0.95 (0.23, 3.89)
12 192 HBeAg Loss 48.0 (12/25) (28.3, 68.2) 66.7 (12/18) (41.2, 85.6) 1.85 (0.45, 7.96)
Anti-HBe Gain 28.0 (7/25) (12.9, 49.6) 77.8 (14/18) (51.9, 92.6) 1.36 (0.27, 7.62)

y
Seroconversion 28.0 (7/25) (12.9, 49.6) 77.8 (14/18) (51.9, 92.6) 1.36 (0.27, 7.62)

op
Histologic not available
Biochemical 56.7 (17/30) (37.7, 74.0) 27.3 (3/11) (7.3, 60.7) 0.49 (0.07, 2.64)
24 192 HBeAg Loss 43.3 (13/30) (26.0, 62.3) 72.7 (8/11) (39.3, 92.7) 2.04 (0.38, 14.07)

C
Anti-HBe Gain 33.3 (10/30) (17.9, 52.9) 100.0 (11/11) (67.9, 100.0) >5.00d
Seroconversion 33.3 (10/30) (17.9, 52.9) 100.0 (11/11) (67.9, 100.0) >5.00d

d
Histologic not available

lle
Biochemical 76.9 (20/26) (55.9, 90.2) 53.3 (8/15) (27.4, 77.7) 3.81 (0.80, 18.49)
48 192 HBeAg Loss 46.2 (12/26) (27.1, 66.3) 60.0 (9/15) (32.9, 82.5) 1.29 (0.30, 5.77)
Anti-HBe Gain 34.6 (9/26) (17.9, 55.6) 86.7 (13/15) (58.4, 97.7) 3.44 (0.55, 37.07)

tro
Seroconversion 34.6 (9/26) (17.9, 55.6) 86.7 (13/15) (58.4, 97.7) 3.44 (0.55, 37.07)
Histologic 100.0 (5/5) (46.3, 100.0) 75.0 (3/4) (21.9, 98.7) *

on
Biochemical 73.7 (14/19) (48.6, 89.9) 33.3 (5/15) (13.0, 61.3) 1.40 (0.25, 7.92)
12 240 HBeAg Loss 57.9 (11/19) (34.0, 78.9) 73.3 (11/15) (44.8, 91.1) 3.78 (0.72, 21.84)
Anti-HBe Gain 27.8 (5/18) (10.7, 53.6) 100.0 (15/15) (74.7, 100.0) >5.38d

C
Seroconversion 26.3 (5/19) (10.1, 51.4) 100.0 (15/15) (74.7, 100.0) >5.00d
Histologic 71.4 (5/7) (30.3, 94.9)
a 100.0 (2/2) (19.8, 100.0) *
Biochemical 69.6 (16/23) (47.0, 85.9) 33.3 (3/9) (9.0, 69.1) 1.14 (0.14, 7.48)
24 240 HBeAg Loss 52.2 (12/23) (31.1, 72.6) 88.9 (8/9) (50.7, 99.4) 8.73 (0.86, 418.80)
ot
Anti-HBe Gain 22.7 (5/22) (8.7, 45.8) 100.0 (9/9) (62.9, 100.0) >2.35d
Seroconversion 21.7 (5/23) (8.3, 44.2) 100.0 (9/9) (62.9, 100.0) >2.22d
-N

Histologic 66.7 (4/6) (24.1, 94.0) 100.0 (2/2) (19.8, 100.0) *

Biochemical 77.3 (17/22) (54.2, 91.3) 40.0 (4/10) (13.7, 72.6) 2.27 (0.32, 14.76)
48 240 HBeAg Loss 54.5 (12/22) (32.7, 74.9) 80.0 (8/10) (44.2, 96.5) 4.80 (0.69, 53.92)
y

Anti-HBe Gain 19.0 (4/21) (6.3, 42.6) 100.0 (10/10) (65.5, 100.0) >2.12d
Seroconversion 18.2 (4/22) (6.0, 41.0) 100.0 (10/10) (65.5, 100.0) >2.00d
nl

* Undefined (division by zero)

O

a
Shading indicates statistical significance.
b
Seroconversion - HBeAg undetectable and antibody against HBeAg detected.
c
An association with, rather than prediction of, clinical responses is demonstrated when measuring the viral response at Week 48.
n

d
The odds ratio calculations are undefined when NPV is 100% or PPV is 100% or missing. Where the denominators for both the NPV and PPV are greater than five, a “minimum” odds ratio was

determined by subtracting one specimen from the numerator of the 100% parameter estimate (NPV or PPV).
io

Tables 30 and 31 demonstrate that the NPV is high (greater than or equal to 97% for response at Week 48) for the association of early viral response with the combination of
at

all three responses (histologic, biochemical, and serological - HBeAg loss or seroconversion). These data indicate that HBeAg-positive subjects without an early viral response
(defined as ≥ 2 log IU/mL decrease) are unlikely to achieve all three clinical responses by Week 48, as a result of treatment. The number of subjects at Week 240 was small, and
therefore available data is inadequate to draw conclusions about the association of the early viral response with clinical responses at this time point.
m

Table 30
or

PPV, NPV, and Odds Ratio (OR) for a Combination of Histologic, Biochemical, and HBeAg Loss Responses During Treatment
nf

Predicted by an Early Viral Response ( ≥ 2 Log IU/mL Decrease) in HBeAg-Positive Subjects

Week of Viral Week of Clinical PPV NPV
rI

PPV (%) (Proportion) NPV (%) (Proportion) Odds Ratio (95% CI)a
Response Response (95% CI) (95% CI)
12 48 23.7 (18/76) (15.0, 35.1) 97.3 (36/37) (84.2, 99.9) 11.17 (1.60, 478.17)
Fo

24 48 20.7 (17/82) (12.9, 31.4) 100.0 (27/27) (84.5, 100.0) >6.80c

48b 48 21.3 (19/89) (13.7, 31.6) 100.0 (31/31) (86.3, 100.0) >8.14c
12 240 60.0 (3/5) (17.0, 92.7) 75.0 (3/4) (21.9, 98.7) 4.50 (0.15, 313.49)
24 240 42.9 (3/7) (11.8, 79.8) 100.0 (2/2) (19.8, 100.0) *
48 240 50.0 (3/6) (13.9, 86.1) 100.0 (2/2) (19.8, 100.0) *
* Undefined (division by zero)
a
Shading indicates statistical significance.
b
An association with, rather than prediction of, clinical responses is demonstrated when measuring the viral response at Week 48.
c
The odds ratio calculations are undefined when NPV is 100% or PPV is 100% or missing. Where the denominators for both the NPV and PPV are greater than five, a “minimum” odds ratio was

determined by subtracting one specimen from the numerator of the 100% parameter estimate (NPV or PPV).

18
 Table 31
PPV, NPV, and Odds Ratio (OR) for a Combination of Histologic, Biochemical, and Seroconversion Responses During Treatment Predicted by an Early Viral
Response ( ≥ 2 Log IU/mL Decrease) in HBeAg-Positive Subjects
Week of Viral Week of Clinical PPV NPV
PPV (%) (Proportion) NPV (%) (Proportion) Odds Ratio (95% CI)
Response Response (95% CI) (95% CI)

 
12 48 10.5 (8/76) (5.0, 20.2) 100.0 (37/37) (88.3, 100.0) >4.24b
24 48 8.5 (7/82) (3.8, 17.3) 100.0 (27/27) (84.5, 100.0) >2.43b
48a 48 9.0 (8/89) (4.2, 17.4) 100.0 (31/31) (86.3, 100.0) >2.96b
12 240 40.0 (2/5) (7.3, 83.0) 100.0 (4/4) (39.6, 100.0) *
24 240 28.6 (2/7) (5.1, 69.7) 100.0 (2/2) (19.8, 100.0) *
48 240 16.7 (1/6) (0.9, 63.5) 100.0 (2/2) (19.8, 100.0) *
* Undefined (division by zero)
a
An association with, rather than prediction of, clinical responses is demonstrated when measuring the viral response at Week 48.
b
The odds ratio calculations are undefined when NPV is 100% or PPV is 100% or missing. Where the denominators for both the NPV and PPV are greater than five, a “minimum” odds ratio was

determined by subtracting one specimen from the numerator of the 100% parameter estimate (NPV or PPV).

HBeAg-Negative Patients
Characterization of Viral Load
Table 32 below demonstrates the efficacy, based on HBV viral load testing, of treating HBeAg-negative patients with 10 mg adefovir dipivoxil compared to placebo. At Week 48,

y
48.72% (57/117) of HBeAg-negative patients on treatment versus 0% (0/55) on placebo had achieved very low viral loads below 100 IU/mL. Furthermore, 3.42% (4/117) of patients

op
on treatment versus 30.91 % (17/55) on placebo had viral loads greater than or equal to 106 IU/mL.

Table 32

C
Distribution of HBV Viral Load at Week 48 for HBeAg-Negative Patients

d
Viral Load Adefovir Dipivoxil Placebo

lle
(IU/mL) N % Cumulative % N % Cumulative %
TNDa 9 7.69 7.69 0 0.00 0.00
< 15 20 17.09 24.79 0 0.00 0.00

tro
15 - < 100 28 23.93 48.72 0 0.00 0.00
100 - < 103 33 28.21 76.92 7 12.73 12.73

on
103 - < 104 9 7.69 84.62 5 9.09 21.82
104 - < 105 6 5.13 89.74 11 20.00 41.82
105 - < 106 8 6.84 96.58 15 27.27 69.09

C
106 - < 109 4 3.42 100.00 17 30.91 100.00
≥ 109 0 0.00 100.00 a 0 0.00 100.00
Total 117 100.00 55 100.00
a
TND = Target Not Detected
ot

Figure 9 below demonstrates the median viral load change and inter-quartile range of change from baseline to Week 48 for HBeAg-negative subjects on treatment compared to
-N

placebo. This shows the impact of adefovir dipivoxil treatment on the viral load of HBeAg-negative patients with chronic hepatitis B.
Figure 9
Median and Inter-Quartile Range of Change in
y

HBV DNA from Baseline: HBeAg-Negative Subjects

nl
O
n
Change in HBV DNA (Log IU/mL)

io
at
m
or
nf
rI

Study Timepoint (WeeK)

Fo

Patients on Placebo
Patients on Treatment

The effect of therapy for patients with chronic HBV infection can be assessed by measuring the HBV DNA (expected reduction to low or undetectable levels) and monitoring for
viral rebound that could be associated with resistance. Results in Table 33 show that 56.91% (70/123) of the treated patients achieved a nadir, or lowest concentration, viral load
level by Week 48. Of the 30 subjects that achieved a nadir by Week 24, 26.92% (7/26) subjects had a greater than or equal to one log IU/mL increase by Week 48 (four of these
subjects did not have a Week 48 result).

19
 Table 33
Distribution of the HBeAg-Negative Subjects by Week on
Treatment and the Viral Load at Which the Nadir was Reached
Nadir Viral Load Number (%) of Patients With the Nadir Viral Load Achieved by Week Total By Viral Cumulative By
(IU/mL) 12 24 48 96 144 192 240 Load Viral Load
0
TNDa 1 (0.81) 3 (2.44) 9 (7.32) 4 (3.25) 11 (8.94) 2 (1.63) 30 (24.39) 30 (24.39)
(0.00)
2
< 15 1 (0.81) 5 (4.07) 6 (4.88) 3 (2.44) 8 (6.50) 8 (6.50) 33 (26.83) 63 (51.22)
(1.63)
2
15 - < 100 2 (1.63) 4 (3.25) 10 (8.13) 1 (0.81) 3 (2.44) 1 (0.81) 23 (18.70) 86 (69.92)
(1.63)
1
100 - < 103 2 (1.63) 3 (2.44) 12 (9.76) 3 (2.44) 1 (0.81) 0 (0.00) 22 (17.89) 108 (87.80)
(0.81)
1
10 - < 10
3 4
2 (1.63) 0 (0.00) 1 (0.81) 0 (0.00) 0 (0.00) 1 (0.81) 5 (4.07) 113 (91.87)
(0.81)
0
10 - < 10
4 5
0 (0.00) 2 (1.63) 1 (0.81) 0 (0.00) 0 (0.00) 0 (0.00) 3 (2.44) 116 (94.31)
(0.00)
0
10 - < 10
5 6
2 (1.63) 1 (0.81) 1 (0.81) 0 (0.00) 1 (0.81) 0 (0.00) 5 (4.07) 121 (98.37)
(0.00)
0
10 - < 10
6 9
1 (0.81) 1 (0.81) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 2 (1.63) 123 (100.00)
(0.00)

y
6

op
Total By Week 11 (8.94) 19 (15.45) 40 (32.52) 11 (8.94) 24 (19.51) 12 (9.76)
(4.88)

Cumulative By Week 11 (8.94) 30 (24.39) 70 (56.91) 81 (65.85) 105 (85.37) 117 (95.12) 123 (100.00)

C
a
TND = Target Not Detected

d
Two patients out of 123 achieved HBsAg seroconversion. One patient was a white female, HBV genotype D, > age 30, and seroconverted at Week 96. The other patient was a

lle
white male, HBV genotype D, > age 30, and seroconverted at Week 240. A summary of these results is provided in Table 34.

tro
Table 34
HBeAg-Negative Subjects with HBsAg Seroconversion

on
Concentration (Log IU/mL)
Week 0 Week 12 Week 24 Week 48 Week 96 Week 144 Week 192 Week 240

C
Subject 1 6.86 3.83 3.25 < 1.18 TND* n/a** n/a** n/a**
Subject 2 6.36 4.53 4.56 5.04 a 5.09 < 1.00 TND* TND*
* TND = Target Not Detected
ot
** Aliquot not available for testing.
-N

Summaries of the effect of baseline covariates for the HBeAg-negative population are provided in Table 35 through Table 37.

Table 35
Association Between Responses to Treatment at Week 48 and Baseline Covariates
y

for HBeAg-Negative Patients

nl

No. of Proportion (%)

O

Response to Patients with of Patients with Unadjusted Odds

Treatment Covariate Category N Response Response Ratio (95% CI)
Asian 32 19 59.38
Race 0.52 (0.20, 1.38)
n

Other 76 56 73.68
io

Male 90 63 70.00
Sex 1.17 (0.32, 3.79)
Female 18 12 66.67
Histological
at

≤ 30 7 6 85.71
Age 2.78 (0.31, 131.93)
> 30 101 69 68.32
B,C 32 19 59.38
m

Genotype 0.52 (0.20, 1.38)

Non-B,C 76 56 73.68
Asian 30 21 70.00
or

Race 0.68 (0.24, 1.99)

Other 80 62 77.50
Male 93 67 72.04
nf

Sex 0.16 (0.00, 1.16)

Female 17 16 94.12
Biochemical
≤ 30 7 6 85.71
rI

Age 2.03 (0.23, 96.72)

> 30 103 77 74.76
B,C 30 21 70.00
Genotype 0.68 (0.24, 1.99)
Fo

Non-B,C 80 62 77.50

The statistical significance of the associations of the Race, Sex, Age and Genotype covariates with viral response was studied by calculating odds ratios and their exact 95%
confidence intervals for both definitions of viral response and summarized in Table 36 and Table 37. All lower limits of the 95% confidence intervals in these two tables are smaller
than 1. This is in concordance with logistic regression analyses of viral response as a function of covariates indicating no statistically significant associations between the four
covariates and viral load. Therefore, the virological responses at Weeks 12, 24 and 48 do not appear to be correlated with Race, Sex, Age, and HBV Genotype.

20
 Table 36
Odds Ratios for the Association Between Viral Response (< 2,000 IU/mL) and Covariates, by Week,
for an HBeAg-Negative Population
No. Below Proportion (%) Unadjusted Odds
Covariate Category Week N 2,000 IU/mL Below 2,000 IU/mL Ratio (95% CI)
Asian 33 25 75.76
12 2.36 (0.89, 6.79)
Other 79 45 56.96
Asian 34 26 76.47
Race 24 1.03 (0.37, 3.08)
Other 79 60 75.95
Asian 33 29 87.88
48 2.34 (0.69, 10.20)
Other 82 62 75.61
Male 94 59 62.77
12 1.07 (0.32, 3.36)
Female 18 11 61.11
Male 93 72 77.42
Sex 24 1.47 (0.41, 4.71)
Female 20 14 70.00
Male 95 76 80.00
48 1.33 (0.34, 4.51)
Female 20 15 75.00
≤ 30 7 4 57.14
12 0.79 (0.13, 5.67)
> 30 105 66 62.86
≤ 30 7 5 71.43
Age 24 0.77 (0.12, 8.59)
> 30 106 81 76.42

y
≤ 30 6 4 66.67

op
48 0.51 (0.07, 5.97)
> 30 109 87 79.82
B,C 33 25 75.76
12 2.36 (0.89, 6.79)
Non-B,C 79 45 56.96

C
B,C 34 26 76.47
Genotype 24 1.03 (0.37, 3.08)
Non-B,C 79 60 75.95

d
B,C 33 29 87.88
48 2.34 (0.69, 10.20)
Non-B,C 82 62 75.61

lle
tro
Table 37
Odds Ratios for the Association Between Viral Response ( ≥ 2 Log Decrease From Baseline Result) and Covariates, by Week,
for an HBeAg-Negative Population

on
No. with ≥ 2 Proportion (%) with Unadjusted Odds
Covariate Category Week N Log Decrease ≥ 2 Log Decrease Ratio (95% CI)

C
Asian 33 25 75.76
12 0.86 (0.30, 2.60)
Other 79 62a 78.48
Asian 34 31 91.18
Race 24 1.50 (0.35, 9.02)
Other 79 69 87.34
ot
Asian 33 31 93.94
48 2.15 (0.42, 21.22)
Other 82 72 87.80
-N

Male 94 72 76.60
12 0.65 (0.11, 2.64)
Female 18 15 83.33
Male 93 82 88.17
Sex 24 0.83 (0.08, 4.32)
Female 20 18 90.00
y

Male 95 83 87.37
48 0.00 (0.00, 1.28)
Female 20 20 100.00
nl

≤ 30 7 5 71.43
12 0.70 (0.11, 7.84)
> 30 105 82 78.10
O

≤ 30 7 6 85.71
Age 24 0.77 (0.08, 38.13)
> 30 106 94 88.68
n

≤ 30 6 5 83.33
48 0.56 (0.06, 28.93)
> 30 109 98 89.91
io

B,C 33 25 75.76
12 0.86 (0.30, 2.60)
Non-B,C 79 62 78.48
at

B,C 34 31 91.18
Genotype 24 1.50 (0.35, 9.02)
Non-B,C 79 69 87.34
m

B,C 33 31 93.94
48 2.15 (0.42, 21.22)
Non-B,C 82 72 87.80
or
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Positive Predictive Value (PPV), Negative Predictive Value (NPV), and Odds Ratio (OR) Analysis in an HBeAg-Negative Population
For each patient, two responses: Histologic and Biochemical - were measured at various times during treatment.
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• Histologic response - improvement of histologic status by at least 2 units of the Knodell necroinflammatory score without deterioration of the fibrosis score compared to the
histologic status at baseline
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• Biochemical response - normalization of ALT test result compared to the biochemical status at the baseline
Additionally, HBsAg seroconversion data were collected. Two patients out of 123 achieved HBsAg seroconversion. One patient was a white female, HBV genotype D, > age 30, and
seroconverted at Week 96. The other patient was a white male, HBV genotype D, > age 30, and seroconverted at Week 240. A summary of these results is provided in Table 34.
Viral load response was defined as either HBV DNA less than 2,000 IU/mL or greater than or equal to 2 log IU/mL decrease from baseline. Statistical analysis (PPV) was
performed to evaluate the association between the clinical responses at Weeks 48, 96, 144, 192, or 240 and a viral load response at Weeks 12, 24, or 48. Statistical analysis
(NPV) was performed to evaluate whether there is an association between the clinical non-responses at Weeks 48, 96, 144, 192, or 240 and a viral load non-response at Weeks
12, 24, or 48.

Viral Response < 2,000 IU/mL

As shown in Table 38, viral response at Weeks 24 and 48 (when defined as < 2,000 IU/mL) appears informative (i.e., lower 95% CI limit for the odds ratio exceeding 1.0) in
predicting biochemical response at 48 and 96 weeks on treatment. The PPV for the association of viral response and histologic response increased throughout the study and
was > 68.3% at Week 48 and > 85.7% at the end of the study. The PPV for the association of viral response and biochemical response was > 78.3% at Week 48 and remained
consistent through the end of the study.

21
 Table 38
PPV, NPV, and Odds Ratio for Individual Clinical Responses During Treatment
Predicted by Early Viral Response (< 2,000 IU/mL) in HBeAg-Negative Subjects
Week Week of
Clinical PPV (%) PPV NPV % NPV
of Viral Clinical Odds Ratio (95% CI)a
Response (Proportion) (95% CI) (Proportion) (95% CI)
Response Response
Histologic 68.3 (41/60) (54.9, 79.4) 29.7 (11/37) (16.4, 47.2) 0.91 (0.34, 2.41)
12 48
Biochemical 78.3 (47/60) (65.5, 87.5) 27.5 (11/40) (15.1, 44.1) 1.37 (0.48, 3.82)
Histologic 71.4 (55/77) (59.8, 80.9) 30.4 (7/23) (14.1, 53.0) 1.09 (0.33, 3.30)
24 48
Biochemical 81.6 (62/76) (70.7, 89.2) 44.0 (11/25) (25.0, 64.7) 3.48 (1.15, 10.28)
Histologic 69.8 (60/86) (58.8, 79.0) 35.0 (7/20) (16.3, 59.1) 1.24 (0.37, 3.82)
48b 48
Biochemical 83.3 (70/84) (73.3, 90.3) 45.8 (11/24) (26.2, 66.8) 4.23 (1.39, 12.62)
Histologic 88.9 (8/9) (50.7, 99.4) 16.7 (1/6) (0.9, 63.5) 1.60 (0.02, 141.06)
12 96
Biochemical 83.3 (25/30) (64.5, 93.7) 32.1 (9/28) (16.6, 52.4) 2.37 (0.59, 10.41)
Histologic 84.6 (11/13) (53.7, 97.3) 33.3 (1/3) (1.8, 87.5) 2.75 (0.03, 78.72)
24 96
Biochemical 87.8 (36/41) (73.0, 95.4) 56.3 (9/16) (30.6, 79.2) 9.26 (1.97, 45.25)
Histologic 85.7 (12/14) (56.2, 97.5) 25.0 (1/4) (1.3, 78.1) 2.00 (0.03, 50.57)
48 96
Biochemical 82.6 (38/46) (68.0, 91.7) 50.0 (7/14) (24.0, 76.0) 4.75 (1.06, 20.85)
Histologic not available
12 144

y
Biochemical 72.7 (24/33) (54.2, 86.1) 30.4 (7/23) (14.1, 53.0) 1.17 (0.30, 4.37)
Histologic not available

op
24 144
Biochemical 76.7 (33/43) (61.0, 87.7) 46.2 (6/13) (20.4, 73.9) 2.83 (0.62, 12.41)
Histologic not available
48 144

C
Biochemical 74.5 (35/47) (59.4, 85.6) 41.7 (5/12) (16.5, 71.4) 2.08 (0.43, 9.30)
Histologic not available
12 192
Biochemical 82.8 (24/29) (63.5, 93.5) 10.0 (2/20) (1.8, 33.1) 0.53 (0.05, 3.78)

d
Histologic not available

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24 192
Biochemical 84.6 (33/39) (68.8, 93.6) 22.2 (2/9) (3.9, 59.8) 1.57 (0.13, 11.50)
Histologic not available
48 192

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Biochemical 86.0 (37/43) (71.4, 94.2) 25.0 (2/8) (4.5, 64.4) 2.06 (0.16, 15.55)
Histologic 85.7 (6/7) (42.0, 99.2) 25.0 (3/12) (6.7, 57.2) 2.00 (0.12, 122.23)
12 240
Biochemical 78.3 (18/23) (55.8, 91.7) 6.3 (1/16) (0.3, 32.3) 0.24 (0.00, 2.58)

on
Histologic 100.0 (12/12) (69.9, 100.0) 42.9 (3/7) (11.8, 79.8) >8.25c
24 240
Biochemical 80.0 (24/30) (60.9, 91.6) 11.1 (1/9) (0.6, 49.3) 0.50 (0.01, 5.31)
Histologic 100.0 (12/12) (69.9, 100.0) 42.9 (3/7) (11.8, 79.8) >8.25c

C
48 240
Biochemical 80.6 (25/31) (61.9, 91.9) 11.1 (1/9) (0.6, 49.3) 0.52 (0.01, 5.51)
a
Shading indicates statistical significance. a
b
An association with, rather than prediction of, clinical responses is demonstrated when measuring the viral response at Week 48.
c
The odds ratio calculations are undefined when NPV is 100% or PPV is 100% or missing. Where the denominators for both the NPV and PPV are greater than five, a “minimum” odds ratio
ot
was determined by subtracting one specimen from the numerator of the 100% parameter estimate (NPV or PPV).
-N

Table 39 shows PPV, NPV, and OR results for a combination of both histologic and biochemical responses. PPV for the association of viral response at 12, 24, and 48 Weeks
(when defined as < 2,000 IU/mL) with the combination of both responses (histologic and biochemical) at Week 240 ranges from 85% to 100% (however, 95% CI is wide and OR
is not significant with the available population).
y

Table 39
nl

PPV, NPV, and Odds Ratio (OR) for a Combination of Histologic and Biochemical Responses During Treatment Predicted by an Early Viral
Response (< 2,000 IU/mL) in HBeAg-Negative Subjects
O

Week of Viral Week of Clinical PPV NPV

PPV (%) (Proportion) NPV (%) (Proportion) Odds Ratio (95% CI)a
Response Response (95% CI) (95% CI)
n

12 48 60.0 (33/55) (45.9, 72.7) 51.4 (19/37) (34.7, 67.8) 1.58 (0.63, 3.99)
io

24 48 64.8 (46/71) (52.5, 75.5) 65.2 (15/23) (42.8, 82.8) 3.45 (1.17, 10.66)
48b 48 62.5 (50/80) (50.9, 72.9) 70.0 (14/20) (45.7, 87.2) 3.89 (1.22, 13.55)
at

12 96 55.6 (5/9) (22.7, 84.7) 50.0 (3/6) (13.9, 86.1) 1.25 (0.10, 15.38)
24 96 61.5 (8/13) (32.3, 84.9) 100.0 (3/3) (31.0, 100.0) *
m

48 96 64.3 (9/14) (35.6, 86.0) 75.0 (3/4) (21.9, 98.7) 5.40 (0.30, 314.24)
12 240 85.7 (6/7) (42.0, 99.2) 25.0 (3/12) (6.7, 57.2) 2.00 (0.12, 122.23)
or

24 240 100.0 (12/12) (69.9, 100.0) 42.9 (3/7) (11.8, 79.8) >8.25c
48 240 100.0 (12/12) (69.9 100.0) 42.9 (3/7) (11.8, 79.8) >8.25c
nf

* Undefined (division by zero)

a
Shading indicates statistical significance.
rI

b
An association with, rather than prediction of, clinical responses is demonstrated when measuring the viral response at Week 48.
c
The odds ratio calculations are undefined when NPV is 100% or PPV is 100% or missing. Where the denominators for both the NPV and PPV are greater than five, a “minimum” odds ratio was
Fo

determined by subtracting one specimen from the numerator of the 100% parameter estimate (NPV or PPV).

Viral Response ≥ 2 Log IU/mL Decrease
As shown in Table 40, viral response at Weeks 12, 24, and 48, when defined as ≥ 2 log decrease in value from the baseline viral load result, is informative (i.e., lower limits of
the 95% CIs exceed 1) in predicting biochemical response at Week 48 of treatment. Viral response at Weeks 12 and 48 is also informative for predicting biochemical response at
Week 96 on treatment. The PPV for the association of viral response at Weeks 12, 24 and 48 and histologic response at the end of the study (240 weeks) was 76.9 %, 86.7%,
and 82.4%, respectively. The PPV for the association of viral response and biochemical response was greater than or equal to 78.8% for biochemical response at Week 48 and
remained consistent through the end of the study.

22
 Table 40
PPV, NPV, and Odds Ratio for Individual Clinical Responses During Treatment
Predicted by Early Viral Response (≥ 2 Log IU/mL Decrease) in HBeAg-Negative Subjects

Week of Viral Week of Clinical PPV (%) PPV NPV (%) NPV
Clinical Response Odds Ratio (95% CI)a
Response Response (Proportion) (95% CI) (Proportion) (95% CI)
Histologic 71.2 (52/73) (59.3, 80.9) 37.5 (9/24) (19.5, 59.2) 1.49 (0.49, 4.30)
12 48
Biochemical 83.1 (64/77) (72.5, 90.4) 47.8 (11/23) (27.4, 68.9) 4.51 (1.44, 13.91)
Histologic 70.8 (63/89) (60.0, 79.7) 27.3 (3/11) (7.3, 60.7) 0.91 (0.14, 4.18)
24 48
Biochemical 78.9 (71/90) (68.8, 86.5) 54.5 (6/11) (24.6, 81.9) 4.48 (1.00, 20.41)
Histologic 68.0 (66/97) (57.7, 76.9) 22.2 (2/9) (3.9, 59.8) 0.61 (0.06, 3.46)
48 b
48
Biochemical 83.3 (80/96) (74.0, 89.9) 75.0 (9/12) (42.8, 93.3) 15.00 (3.17, 92.12)
Histologic 83.3 (10/12) (50.9, 97.1) 0.0 (0/3) (0.0, 69.0) 0.0 (0.00, 15.31)
12 96
Biochemical 84.1 (37/44) (69.3, 92.8) 50.0 (7/14) (24.0, 76.0) 5.29 (1.14, 24.06)
Histologic 84.6 (11/13) (53.7, 97.3) 33.3 (1/3) (1.8, 87.5) 2.75 (0.03, 78.72)
24 96
Biochemical 81.3 (39/48) (66.9, 90.6) 55.6 (5/9) (22.7, 84.7) 5.42 (0.92, 32.22)
Histologic 82.4 (14/17) (55.8, 95.3) 0.0 (0/1) (0.0, 94.5) 0.00 (0.00, 95.00)
48 96
Biochemical 80.0 (44/55) (66.6, 89.1) 80.0 (4/5) (29.9, 98.9) 16.00 (1.32, 805.02)
Histologic not available
12 144
Biochemical 75.0 (33/44) (59.4, 86.3) 41.7 (5/12) (16.5, 71.4) 2.14 (0.44, 9.72)

y
Histologic not available

op
24 144
Biochemical 72.9 (35/48) (57.9, 84.3) 37.5 (3/8) (10.2, 74.1) 1.62 (0.22, 9.67)
Histologic not available
48 144

C
Biochemical 72.7 (40/55) (58.8, 83.5) 50.0 (2/4) (9.2, 90.8) 2.67 (0.18, 39.14)
Histologic not available
12 192
Biochemical 89.7 (35/39) (74.8, 96.7) 30.0 (3/10) (8.1, 64.6) 3.75 (0.44, 27.27)

d
Histologic not available
24 192

lle
Biochemical 85.4 (35/41) (70.1, 93.9) 28.6 (2/7) (5.1, 69.7) 2.33 (0.18, 18.75)
Histologic not available
48 192

tro
Biochemical 87.5 (42/48) (74.1, 94.8) 66.7 (2/3) (12.5, 98.2) 14.00 (0.59, 844.47)
Histologic 76.9 (10/13) (46.0, 93.8) 16.7 (1/6) (0.9, 63.5) 0.67 (0.01, 11.40)
12 240
Biochemical 82.8 (24/29) (63.5, 93.5) 10.0 (1/10) (0.5, 45.9) 0.53 (0.01, 5.90)

on
Histologic 86.7 (13/15) (58.4, 97.7) 25.0 (1/4) (1.3, 78.1) 2.17 (0.03, 54.35)
24 240
Biochemical 78.8 (26/33) (60.6, 90.4) 0.0 (0/6) (0.0, 48.3) 0.00 (0.00, 2.99)
Histologic 82.4 (14/17) (55.8, 95.3) 0.0 (0/2) (0.0, 80.2) 0.00 (0.00, 20.71)

C
48 240
Biochemical 81.6 (31/38) (65.1, 91.7) 0.0 (0/2) (0.0, 80.2) 0.00 (0.00, 17.04)
a
Shading indicates statistical significance. a
b
An association with, rather than prediction of, clinical responses is demonstrated when measuring the viral response at Week 48.
ot
Table 41 shows PPV, NPV, and OR results for a combination of both histologic and biochemical responses. PPV for the association of viral response at 12, 24, and 48 weeks (when
defined as ≥ 2 log IU/mL decrease in value from the baseline viral load result) with the combination of both responses (histologic and biochemical) at Week 240 ranges from
76.9% to 86.7% (however, 95% CI is wide, and OR is not significant with the available population).
-N

Table 41
PPV, NPV, and Odds Ratio (OR) for a Combination of Histologic and Biochemical Responses During Treatment Predicted by an Early Viral
y

Response (≥ 2 Log IU/mL Decrease) in HBeAg-Negative Subjects

nl

Week of Viral Week of Clinical PPV (%) PPV NPV (%) NPV
Odds Ratio (95% CI)a
O

Response Response (Proportion) (95% CI) (Proportion) (95% CI)

12 48 61.4 (43/70) (49.0, 72.6) 63.6 (14/22) (40.8, 82.0) 2.79 (0.93, 8.68)
24 48 59.5 (50/84) (48.2, 69.9) 60.0 (6/10) (27.4, 86.3) 2.21 (0.48, 11.36)
n

48b 48 60.4 (55/91) (49.6, 70.4) 88.9 (8/9) (50.7, 99.4) 12.22 (1.50, 551.92)
io

12 96 58.3 (7/12) (28.6, 83.5) 66.7 (2/3) (12.5, 98.2) 2.80 (0.11, 188.36)
24 96 53.8 (7/13) (26.1, 79.6) 66.7 (2/3) (12.5, 98.2) 2.33 (0.09, 157.00)
at

48 96 58.8 (10/17) (33.5, 80.6) 100.0 (1/1) (5.5, 100.0) *

12 240 76.9 (10/13) (46.0, 93.8) 16.7 (1/6) (0.9, 63.5) 0.67 (0.01, 11.40)
m

24 240 86.7 (13/15) (58.4, 97.7) 25.0 (1/4) (1.3, 78.1) 2.17 (0.03, 54.35)
or

48 240 82.4 (14/17) (55.8, 95.3) 0.0 (0/2) (0.0, 80.2) 0.00 (0.00, 20.71)
a
Shading indicates statistical significance.
nf

b
An association with, rather than prediction of, clinical responses is demonstrated when measuring the viral response at Week 48.
* Undefined (division by zero)
rI

Conclusions Drawn from the Studies

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Based on the results of the nonclinical and clinical laboratory studies, the Abbott RealTime HBV assay, when used according to the provided directions and in conjunction with
other serological and clinical information, is found to be useful in quantitation of Hepatitis B Virus (HBV) DNA in human serum or plasma from chronically HBV-infected individuals,
for aiding in assessing response to treatment and the management of patients with chronic HBV infection undergoing anti-viral therapy by measuring HBV DNA levels at baseline
and during treatment.

23
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C
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tro

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on
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ot
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y

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nl

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io

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at

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m

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or

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nf

Gastroenterology. 2006;131(6):1743–51.
rI

Abbott RealTime HBV is covered by US Patent No. 7,015,317, assigned to Abbott

Laboratories.
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Abbott m, m2000, m2000sp and m2000rt are trademarks of Abbott Laboratories.

ProClin is a registered trademark of Rohm and Haas.
FAM and ROX are trademarks of Life Technologies Corporation or its subsidiaries in the
US and/or certain other countries.
VIC is a registered trademark of Life Technologies Corporation or its subsidiaries in the
US and/or certain other countries.
Abbott Molecular Inc. is the legal manufacturer of the:
Abbott RealTime HBV Amplification Reagent Kit (List No. 2N40-90);
Abbott RealTime HBV Control Kit (List No. 2N40-80); and
Abbott RealTime HBV Calibrator Kit (List No. 2N40-70).
Abbott Proteinase K (List No. 03L78-061)

Abbott Molecular Inc. www.abbottmolecular.com

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Des Plaines, IL 60018 USA June 2016

24